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.     

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.     

ProSAP/Shank proteins - a family of higher order organizing molecules of the postsynaptic density with an emerging role in human neurological disease

The postsynaptic density (PSD) is a specialized electron-dense structure underneath the postsynaptic plasmamembrane of excitatory synapses. It is thought to anchor and cluster glutamate receptors exactly opposite to the presynaptic neurotransmitter release site. Various efforts to study the molecular structure of the PSD identified several new proteins including membrane receptors, cell adhesion molecules, components of signalling cascades, cytoskeletal elements and adaptor proteins with scaffolding functions to interconnect these PSD components. The characterization of a novel adaptor protein family, the ProSAPs or Shanks, sheds new light on the basic structural organization of the PSD. ProSAPs/Shanks are multidomain proteins that interact directly or indirectly with receptors of the postsynaptic membrane including NMDA-type and metabotropic glutamate receptors, and the actin-based cytoskeleton. These interactions suggest that ProSAP/Shanks may be important scaffolding molecules of the PSD with a crucial role in the assembly of the PSD during synaptogenesis, in synaptic plasticity and in the regulation of dendritic spine morphology. Moreover the analysis of a patient with 22q13.3 distal deletion syndrome revealed a balanced translocation with a breakpoint in the human ProSAP2/Shank3 gene. This ProSAP2/Shank3 haploinsufficiency may cause a syndrome that is characterized by severe expressive language delay, mild mental retardation and minor facial dysmorphisms.     

Phosphorylation state of postsynaptic density proteins

The postsynaptic density (PSD) is an electron-dense structure located at the synaptic contacts between neurons. Its considerable complexity includes cytoskeletal and scaffold proteins, receptors, ion channels and signaling molecules, in line with the role of PSDs in signal transduction and processing. The phosphorylation state of components of the PSD is central to synaptic transmission and is known to play a role in synaptic plasticity, learning and memory. The presence of a range of kinases and phosphatases in the PSD defines potential key players in this context. However, the substrates that these enzymes target have not been fully identified to date. We analyzed the protein composition of purified PSD samples from adult mouse brains by strong cation exchange chromatography fractionation of a tryptic digest followed by nano-reverse phase liquid chromatography coupled with electrospray ionization-quadrupole time of flight tandem mass spectrometry. This led to the identification of 244 proteins. To gain an insight into the phosphoproteome of the PSD we then purified phosphorylated tryptic peptides by immobilized metal ion affinity chromatography. This approach for the specific enrichment of phosphopeptides resulted in the identification of 42 phosphoproteins in the PSD preparation, 39 of which are known PSD components. Here we present a total of 83 in vivo phosphorylation sites.     

The synaptic scaffold protein MPP2 interacts with GABAA receptors at the periphery of the postsynaptic density of glutamatergic synapses

Recent advances in imaging technology have highlighted that scaffold proteins and receptors are arranged in subsynaptic nanodomains. The synaptic membrane-associated guanylate kinase (MAGUK) scaffold protein membrane protein palmitoylated 2 (MPP2) is a component of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor–associated protein complexes and also binds to the synaptic cell adhesion molecule SynCAM 1. Using superresolution imaging, we show that—like SynCAM 1—MPP2 is situated at the periphery of the postsynaptic density (PSD). In order to explore MPP2-associated protein complexes, we used a quantitative comparative proteomics approach and identified multiple γ-aminobutyric acid (GABA)_A receptor subunits among novel synaptic MPP2 interactors. In line with a scaffold function for MPP2 in the assembly and/or modulation of intact GABA_A receptors, manipulating MPP2 expression had effects on inhibitory synaptic transmission. We further show that GABA_A receptors are found together with MPP2 in a subset of dendritic spines and thus highlight MPP2 as a scaffold that serves as an adaptor molecule, linking peripheral synaptic elements critical for inhibitory regulation to central structures at the PSD of glutamatergic synapses.

This study shows that the MAGUK scaffold protein MPP2 is located at the periphery of postsynaptic densities in excitatory neurons, where it interacts with GABA-A receptors, thereby serving as a functional adaptor that links excitatory and inhibitory components of synaptic transmission at glutamatergic synapses.     

Coupling of mGluR/Homer and PSD-95 complexes by the Shank family of postsynaptic density proteins.

Shank is a recently described family of postsynaptic proteins that function as part of the NMDA receptor-associated PSD-95 complex (Naisbitt et al., 1999 [this issue of Neuron]). Here, we report that Shank proteins also bind to Homer. Homer proteins form multivalent complexes that bind proline-rich motifs in group 1 metabotropic glutamate receptors and inositol trisphosphate receptors, thereby coupling these receptors in a signaling complex. A single Homer-binding site is identified in Shank, and Shank and Homer coimmunoprecipitate from brain and colocalize at postsynaptic densities. Moreover, Shank clusters mGluR5 in heterologous cells in the presence of Homer and mediates the coclustering of Homer with PSD-95/GKAP. Thus, Shank may cross-link Homer and PSD-95 complexes in the PSD and play a role in the signaling mechanisms of both mGluRs and NMDA receptors.     

Trafficking and synaptic anchoring of ionotropic inhibitory neurotransmitter receptors

Neurotransmitter receptors are subject to microtubule-based transport between intracellular organelles and the neuronal plasma membrane. Receptors that arrive at plasma membrane compartments diffuse laterally within the plane of the cellular surface. To achieve immobilization at their sites of action, cytoplasmic receptor residues bind to submembrane proteins, which are coupled to the underlying cytoskeleton by multiprotein scaffolds. GABA(A)Rs (gamma-aminobutyric type A receptors) and GlyRs (glycine receptors) are the major inhibitory receptors in the central nervous system. At inhibitory postsynaptic sites, all GlyRs and the majority of GABA(A)Rs directly or indirectly couple to gephyrin, a multimeric PSD (postsynaptic density) component. In addition to cluster formations at axo-dendritic contacts, individual GABA(A)R subtypes also anchor and concentrate at extrasynaptic positions, either through association with gephyrin or direct interaction with the ERM (ezrin/radixin/moesin) family protein radixin. In addition to their role in diffusion trapping of surface receptors, scaffold components also undergo rapid exchange to/from and between postsynaptic specializations, leading to a dynamic equilibrium of receptor-scaffold complexes. Moreover, scaffold components serve as adaptor proteins that mediate specificity in intracellular transport complexes. In the present review, we discuss the dynamic delivery, stabilization and removal of inhibitory receptors at synaptic sites.     

Physiology of synapse: from molecular modules to retrograde modulation

Synapses are highly organized, specific structures assuring rapid and highly selective interactions between cells. Synaptic transmission involves the release of neurotransmitter from presynaptic neurons and its detection by specific ligand-gated ion channels at the surface membrane of postsynaptic neurons. The protenomic analysis shows that for self-formation and functioning of synapses nearly 2000 proteins are involved in mammalian brain. The core complex in excitatory synapses includes glutamate receptors, potassium channels, CaMKII, scaffolding protein and actin. These proteins exist as part of a highly organized protein complex known as the postsynaptic density (PSD). The coordinated functioning of the different PSD components determines the strength of signalling between the pre- and postsynaptic neurons. Synaptic plasticity is regulated by changes in the amount of receptors on the postsynaptic membrane, changes in the shape and size of dendritic spines, posttranslational modification of PSD components, modulation kinetics of synthesis and degradation of proteins. Integration of these processes leads to long-lasting changes in synaptic function and neuronal networks underlying learning-related plasticity, memory and information treatment in nervous system of multicellular organisms.     

Semiquantitative proteomic analysis of rat forebrain postsynaptic density fractions by mass spectrometry

The postsynaptic density (PSD) of central excitatory synapses plays a key role in postsynaptic signal transduction and contains a high concentration of glutamate receptors and associated scaffold and signaling proteins. We report here a comprehensive analysis of purified PSD fractions by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We identified 374 different proteins that copurified with the PSD structure and discovered thirteen phosphorylated sites from eight proteins. These proteins were classified into numerous functional groups, implying that the signaling pathways in the PSD are complex and diverse. Furthermore, using quantitative mass spectrometry, we measured the molar concentration and relative stoichiometries of a number of glutamate receptor subunits and scaffold proteins in the postsynaptic density. Thus this proteomic study reveals crucial information about molecular abundance as well as molecular diversity in the PSD, and provides a basis for further studies on the molecular mechanisms of synaptic function and plasticity.     

A Dynamic Corral Model of Receptor Trafficking at a Synapse

The postsynaptic density (PSD) is a cytoskeletal specialization within the postsynaptic membrane of a neuron that helps to concentrate and organize neurotransmitter receptors at a chemical synapse. The total number of receptors within the PSD, which is a major factor in determining the physiological strength or weight of a synapse, fluctuates due to the surface diffusion of receptors into and out of the PSD, and the interactions of receptors with scaffolding proteins and cytoskeletal elements within the PSD. In this article, we present a stochastic model of protein receptor trafficking at the PSD that takes into account these various processes. The PSD is treated as a stochastically gated corral, which contributes a source of extrinsic or environmental noise that supplements the intrinsic noise arising from small receptor numbers. Using a combination of stochastic analysis and Monte Carlo simulations, we determine the time-dependent variation in the mean and variance of synaptic receptor numbers for a variety of initial conditions that simulate fluorescence recovery after photobleaching experiments, and indicate how such data might be used to infer certain properties of the PSD.     

Glutamatergic Postsynaptic Density Protein Dysfunctions in Synaptic Plasticity and Dendritic Spines Morphology: Relevance to Schizophrenia and Other Behavioral Disorders Pathophysiology,and Implications for Novel Therapeutic Approaches

Emerging researches point to a relevant role of postsynaptic density (PSD) proteins, such as PSD-95, Homer, Shank, and DISC-1, in the pathophysiology of schizophrenia and autism spectrum disorders. The PSD is a thickness, detectable at electronic microscopy, localized at the postsynaptic membrane of glutamatergic synapses, and made by scaffolding proteins, receptors, and effector proteins; it is considered a structural and functional crossroad where multiple neurotransmitter systems converge, including the dopaminergic, serotonergic, and glutamatergic ones, which are all implicated in the pathophysiology of psychosis. Decreased PSD-95 protein levels have been reported in postmortem brains of schizophrenia patients. Variants of Homer1, a key PSD protein for glutamate signaling, have been associated with schizophrenia symptoms severity and therapeutic response. Mutations in Shank gene have been recognized in autism spectrum disorder patients, as well as reported to be associated to behaviors reminiscent of schizophrenia symptoms when expressed in genetically engineered mice. Here, we provide a critical appraisal of PSD proteins role in the pathophysiology of schizophrenia and autism spectrum disorders. Then, we discuss how antipsychotics may affect PSD proteins in brain regions relevant to psychosis pathophysiology, possibly by controlling synaptic plasticity and dendritic spine rearrangements through the modulation of glutamate-related targets. We finally provide a framework that may explain how PSD proteins might be useful candidates to develop new therapeutic approaches for schizophrenia and related disorders in which there is a need for new biological treatments, especially against some symptom domains, such as negative symptoms, that are poorly affected by current antipsychotics.     

Neuronal cotransport of glycine receptor and the scaffold protein gephyrin

The dynamics of postsynaptic receptor scaffold formation and remodeling at inhibitory synapses remain largely unknown. Gephyrin, which is a multimeric scaffold protein, interacts with cytoskeletal elements and stabilizes glycine receptors (GlyRs) and individual subtypes of gamma-aminobutyric acid A receptors at inhibitory postsynaptic sites. We report intracellular mobility of gephyrin transports packets over time. Gephyrin units enter and exit active synapses within several minutes. In addition to previous reports of GlyR-gephyrin interactions at plasma membranes, we show cosedimentation and coimmunoprecipitation of both proteins from vesicular fractions. Moreover, GlyR and gephyrin are cotransported within neuronal dendrites and further coimmunoprecipitate and colocalize with the dynein motor complex. As a result, the blockade of dynein function or dynein-gephyrin interaction, as well as the depolymerization of microtubules, interferes with retrograde gephyrin recruitment. Our data suggest a GlyR-gephyrin-dynein transport complex and support the concept that gephyrin-motor interactions contribute to the dynamic and activity-dependent rearrangement of postsynaptic GlyRs, a process thought to underlie the regulation of 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.     

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.     

Scaffolding Proteins of the Post-synaptic Density Contribute to Synaptic Plasticity by Regulating Receptor Localization and Distribution: Relevance for Neuropsychiatric Diseases

Synaptic plasticity represents the long lasting activity-related strengthening or weakening of synaptic transmission, whose well-characterized types are the long term potentiation and depression. Despite this classical definition, however, the molecular mechanisms by which synaptic plasticity may occur appear to be extremely complex and various. The post-synaptic density (PSD) of glutamatergic synapses is a major site for synaptic plasticity processes and alterations of PSD members have been recently implicated in neuropsychiatric diseases where an impairment of synaptic plasticity has also been reported. Among PSD members, scaffolding proteins have been demonstrated to bridge surface receptors with their intracellular effectors and to regulate receptors distribution and localization both at surface membranes and within the PSD. This review will focus on the molecular physiology and pathophysiology of synaptic plasticity processes, which are tuned by scaffolding PSD proteins and their close related partners, through the modulation of receptor localization and distribution at post-synaptic sites. We suggest that, by regulating both the compartmentalization of receptors along surface membrane and their degradation as well as by modulating receptor trafficking into the PSD, postsynaptic scaffolding proteins may contribute to form distinct signaling micro-domains, whose efficacy in transmitting synaptic signals depends on the dynamic stability of the scaffold, which in turn is provided by relative amounts and post-translational modifications of scaffolding members. The putative relevance for neuropsychiatric diseases and possible pathophysiological mechanisms are discussed in the last part of this work.     

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.     

Quantitative analysis of synaptic phosphorylation and protein expression

The postsynaptic density (PSD) signaling machinery contains proteins with diverse functions. Brain region-specific variations in PSD components mediate distinct physiological responses to synaptic activation. We have developed mass spectrometry-based methods to comprehensively compare both relative protein expression and phosphorylation status from proteins present in biochemical preparations of postsynaptic density. Using these methods, we determined the relative expression of 2159 proteins and 1564 phosphorylation sites in PSD preparations from murine cortex, midbrain, cerebellum, and hippocampus. These experiments were conducted twice using independent biological replicates, which allowed us to assess the experimental and biological variability in this system. Concerning protein expression, cluster analysis revealed that known functionally associated proteins display coordinated synaptic expression. Therefore, proteins identified as co-clustering with known protein complexes are prime candidates for assignment as previously unrecognized components. Concerning degree of phosphorylation, we observed more extensive phosphorylation sites on N-methyl-D-aspartate (NMDA) receptors than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, consistent with the central role of N-methyl-D-aspartate receptors in processing synaptic transmission patterns. Average kinase and phosphatase levels were highest in the hippocampus, correlating with a higher overall phosphopeptide abundance present in this brain region. These findings suggest that the hippocampus utilizes reversible protein phosphorylation to a greater extent than other brain regions when modifying synaptic strength.     

The postsynaptic spectrin/4.1 membrane protein "accumulation machine"

An important aspect of the function of the membrane-associated cytoskeleton has been suggested to be to trap and retain selected transmembrane proteins at points on the cell surface specified by cell adhesion molecules. In the process, cell adhesion molecules are cross-linked to each other, and so junctional complexes are strengthened. In this short review, we will discuss recent advances in understanding the role of this "accumulation machine" in postsynaptic structures. Function in the brain depends on correct ordering of synaptic intercellular junctions, and in particular the recruitment of receptors and other apparatus of the signalling system to postsynaptic membranes. Spectrin has long been known to be a component of postsynaptic densities, and recent advances in molecular cloning indicate that beta spectrins at PSDs are all "long" C-terminal isoforms characterised by pleckstrin homology domains. Isoforms of protein 4.1 are also present at synapses. All four 4.1 proteins are represented in PSD preparations, but it is 4.1R that is most enriched in PSDs. 4.1R binds to several proteins enriched in PSDs, including the characteristic PSD intermediate filament, alpha-internexin. Both 4.1 and spectrin interact with ionotropic glutamate receptors (AMPA and NMDA receptors, respectively): 4.1 stabilises AMPA receptors on the cell surface. By linking these receptors to the cytoskeletal and cell adhesion molecules that specify glutamatergic synapses, the membrane protein accumulation machine is suggested to direct the formation of postsynaptic signalling complexes.