Mol Neurobiol DOI 10.1007/s12035-016-9745-1

Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95 Daniela Vallejo 1 & Juan F. Codocedo 1 & Nibaldo C. Inestrosa 1,2,3

Received: 20 October 2015 / Accepted: 22 January 2016 # Springer Science+Business Media New York 2016

Abstract 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

* Nibaldo C. Inestrosa [email protected]

1

Centro de Envejecimiento y Regeneración (CARE), Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, P. O. Box 114-D, Santiago, Chile

2

Centre for Healthy Brain Ageing, School of Psychiatry, Faculty of Medicine, University of New South Wales, Sydney, Australia

3

Centro de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, Punta Arenas, Chile

synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes. Keywords PSD-95 . Postsynaptic density . Posttranslational modifications . Neurodegeneration

Introduction The postsynaptic density (PSD) is a dynamic electron-dense thickened structure located beneath the postsynaptic membrane, whose morphology and protein composition change with neural activity [1]. Different molecules belonging to the family of membrane-associated guanylate kinase (MAGUK) proteins, which have different functions depending on their location, form the PSD [2, 3]. Several functions are attributed to the PSD, including facilitation of membrane receptor anchorage in the dendritic spines, modulation of the trafficking, and localization of adhesion molecules, such as Neuroligin 1 (NL-1), and functional proteins, such as ion channels (Shakertype K+ channels), N-methyl-D-aspartate (NMDA), and αamino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors [4, 5]. Given that the architecture of the PSD controls the amplitude and variance of postsynaptic currents and that its dynamic structure is composed of individual components that are continuously being repositioned [6, 7] and exchanged [8, 9], the organization of the PSD regulates the strength and plasticity of excitatory synaptic neurotransmission. The postsynaptic density protein-95 (PSD-95) resides within the PSD and is closely associated with the membrane receptors and ion channels. This protein is also known as SAP-90 and disc large (Dlg) 4, and it is the most abundant scaffold protein of the dendritic spines [10]. PSD-95 is directly

Mol Neurobiol

involved in organizing the PSD due to its position into vertical filaments that interact with horizontal elements and glutamatergic receptors situated deep inside the PSD [11]. Moreover, immunogold analysis of PSD-95 revealed that it is principally associated with the postsynaptic side: it shows asymmetrical localization at the synaptic junctions of the forebrain [12]. By interacting with various glutamatergic receptors, cell adhesion molecules, and cytoskeletal elements, PSD-95 regulates the structural organization of the PSD [13–15], which is fundamental for synapse development and plasticity. For example, disruption of Dlg, a Drosophila MAGUK protein, alters postsynaptic structure by blocking the clustering of Shaker-type K+ channels that are normally associated with Dlg [16]. In cultured hippocampal neurons, overexpression of PSD-95 accelerates the development of excitatory synapses and selectively enhances the clustering of AMPA receptors (AMPARs) [17]. Furthermore, targeted disruption of PSD-95 causes severe abnormalities in synaptic plasticity, including enhancement of long-term potentiation (LTP) and impairment of long-term depression (LTD). These abnormalities in synaptic plasticity probably explain why PSD-95 mutant mice show impairment in spatial learning tasks [18]. For this reason, it is not surprising that several signaling pathways that regulate synaptic physiology operate by modulating PSD-95 [19, 20]. For example, the activation of the noncanonical Wnt pathway induces increases in dendritic spine density and activity [21]. In fact, aspects of these effects are due to the concomitant increase in PSD-95 clustering mediated by the activation of Jun N-terminal kinase (JNK) [22]. PSD-95 is highly susceptible to modification, mainly due to its conformational structure composed of independent modular domains. In this review, we summarize the structural features of PSD-95 that allow posttranslational modifications to modulate its postsynaptic localization within the dendritic spine, thereby influencing chemical synaptic transmission regulation in the central nervous system (CNS).

Gene Expression, Structural Features, and Main Functions of PSD-95 PSD-95, encoded by Dlg4, is a member of the MAGUK family of synaptic proteins comprising PSD-95, PSD-93, SAP102, and SAP-97. Dlg4 is located on chromosome band 17p13.1 in humans [23], encompasses approximately 30 kb, and is composed of 22 exons [24]. The expression of PSD-95 has been reported to depend on the synaptic activation received; in fact, increased PSD-95 expression is observed in neurons that receive synaptic stimuli [25]. In addition, it has been reported that NeuroD2, a transcription factor that is activated in response to increased intracellular calcium, upregulates PSD-95 expression in hippocampal neurons

[26]. Epigenetic mechanisms are also involved in the regulation of the genetic expression of PSD-95 through the interaction of the transcription factor Neuregulin-1 with histonemodifying enzymes; it has been demonstrated that the histone deacetylase HDAC2 binds to the PSD-95 promoter and regulates its expression [25, 27]. Posttranscriptional factors are also involved in the regulation of PSD-95 expression, including miRNAs such as miR-125a [28]. Furthermore, although PSD-95 messenger RNA (mRNA) is expressed during early development, this mRNA is not translated into protein. Therefore, the expression of PSD-95 is controlled and restricted by at least two mechanisms: the action of miR-125a and the degradation of PSD-95 mRNA, the latter of which is mediated by two polypyrimidine tract binding (PTB) proteins. The overall result is inhibition of excitatory synapse formation as well as neuronal maturation [29]. All the MAGUKs are known hub proteins that are expressed in various submembrane domains. Interestingly, a recent study has shown that MAGUKs are specifically involved in the creation of functional synapses after initial spine formation [30]. Their molecular organization is similar, with conformationally independent modular domains that are connected in series by flexible polypeptide linkers; these domains include three conserved PDZ domains (PSD-95, Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 proteins (zo-1)) and one Src homology 3-guanylate kinase (SH3-GK) module [31, 32]. However, in PSD-95, these domains were more recently classified into two independent supramodules: PDZ1-PDZ2 and PDZ3-SH3-GK [33]. An αhelical segment connects the PDZ3 domain to the subsequent SH3 domain, placing the PDZ3 domain on a different face of the binding pocket [34] (Fig. 1). The PDZ domains are modular protein interaction domains that are mostly specialized for binding to molecular elements present in glutamate receptors or signaling molecules via a Cterminal binding sequence (tSXV), Thr/Ser-X-Val/Ile-COOH) [35]. In addition, these PDZ domains of PSD-95, particularly PDZ1 and PDZ2, are distributed according to a specific distance and tend to bind to pairs of C termini extending in the same direction [36]. Although binding to C-terminal peptides seems to be the most common interaction, PDZ domains can also interact with internal peptide sequences (PDZ-PDZ interactions) [37] due to their capacity to adopt multiple conformational states [38, 39]. This high level of conformational plasticity is achieved mainly through the interdomain sequences [40]; in fact, the multiplicity of conformational states of these multimodular proteins is described as “supertertiary structure” [41]. These multiple PSD-95 domains contribute to stabilizing the PSD of the dendritic spines and to organizing its macromolecular components [9]. Moreover, it is worthy to mention that most of the vertical filaments, the most abundant structural entity in the PSD, contribute in the orientation of the PSD-

Mol Neurobiol

Fig. 1 The conformationally independent modular PDZ domains of PSD-95 and their interactions with other molecules and structures. The figure presents the molecular organization of PSD-95. The scaffold protein is composed of three independent PDZ domains (PDZ1, PDZ2, PDZ3) and the SH3-GK module. These PDZ domains allow PSD-95 to

interact with various molecules and/or structures through its C-terminal binding sequence or via internal peptide sequences such as PDZ-PDZ interactions. The illustration depicts several glutamate receptors, proteins, ion channels, and signaling enzymes that show preferential binding to specific PDZ domains

95 molecules, forming an array of vertically oriented, membrane-associated filaments that label for PSD-95 [42]. For instance, it has been reported that NMDARs bind to PSD-95 through canonical interactions of the GluN2 subunits (NR2A and NR2B) with PDZ1 and PDZ2 [14, 43]; however, the NR2B subunit is occasionally able to interact with the truncated PDZ3 domain [44]. Interestingly, it has been observed that NR2A-NR2C subunits are more strongly bound by the PDZ2 domain compared with other domains [45]. AMPARs are situated at synapses where they form complexes with transmembrane AMPA receptor regulatory proteins (TARPs) like Stargazin, which is the most representative TARP [46, 47]. TARP-containing AMPARs are stabilized at the synapse through the involvement in multivalent interactions with the PDZ domains of PSD-95 [48]. Regarding the interaction of AMPAR-Stargazin complex with PSD-95, although it was initially reported to be preferentially bound by the first two PSD-95 PDZ domains [49, 50], a recent study has revealed that the Stargazin C-tail could be modified, modulating the charge of its serine-rich regions, which determine the extension of the C-tail domain into the cytoplasm. This extension makes the PSD-95 PDZ2/3 domains more accessible for binding, thus promoting the recruitment of additional AMPARs and potentiating chemical synaptic transmission [51]. In addition, it has been suggested that the effective length of the Stargazin C-tail may be central to synaptic transmission [52, 53]. However, the binding of Stargazin to PSD-95 can be inhibited by the interaction of Stargazin with negatively charged lipid bilayers in a phosphorylation-dependent manner; thus, this phosphorylation plays a fundamental role in the

regulation of AMPAR-mediated synaptic transmission [54]. Curiously, it has been reported that PSD-95 domains are preferentially enriched with AMPARs compared with NMDARs [55]. The vertical orientation of PSD-95 molecules aforementioned is responsible of putting its PDZ domains near the membrane in a specific position to bind to other molecules and structures [42]. Other receptor-related PSD structures have been described to interact with different PDZ domains: the receptor tyrosine-protein kinase ErbB4 binds to PDZ1 and PDZ2 [56, 57], GluR6 binds to PDZ1, and the kainate receptor KA2 binds preferentially to the SH3 and GK domains [58] (Fig. 1). NL-1, a cell adhesion protein on the postsynaptic membrane, mediates the formation and maintenance of synapses between neurons. It binds to the PDZ3 domain of PSD-95 through its cytoplasmic C-terminal domain, which contains a PDZ-binding motif [13, 59]. Similarly, it has been reported that the cysteine-rich PDZ-binding protein (CRIPT) [60, 61] also binds to the PDZ3 domain. Moreover, a study has revealed that one of the disintegrin and metalloproteinase domain-containing protein 22 (ADAM22) splicing variants shows preference for the C-terminal half containing the PDZ3 domain of PSD-95 [62]. On the other hand, it has been reported that the PDZ1 and PDZ2 domains of PSD-95 bind to the C-terminal peptide sequence of the Shaker [63] and inward-rectifier K+ channels [64]. Furthermore, the intracellular protein cypin (cytoplasmic PSD-95 interactor) is a PSD-95 binding protein, which decreases the PSD-95 family member localization, regulates dendrite patterning, and possesses a

Mol Neurobiol

PDZ-interacting sequence –SSSV* that binds to PDZ1 and PDZ2 [65, 66]. Regarding signaling enzymes, the internal peptide sequences of neural nitric oxide synthase (nNOS) bind selectively to the PDZ2 domain of PSD-95 [37, 67], whereas Ca2+/calmodulin-dependent protein kinase II (CamKII) tends to bind to PDZ1 [68]. Src family kinases, which are membrane-associated nonreceptor tyrosine kinases, interact with the PDZ3 domain of PSD-95 via their SH2 domains [69], as does stress-activated protein kinase-3 (SAPK3/ p38γ) [70]. Two of the best-characterized binding partners of the PSD-95 SH3-GK are the guanylate kinase-associated protein (GKAP; also known as SAPAP) [15, 71], which participates in facilitating the assembly of the PSD of neurons [72], and MAP1A [73], which plays a role in the microtubule cytoskeleton remodeling [74]. However, the synaptic Ras GTPase-activating protein (SynGAP), an important postsynaptic component and an essential protein in the development of cognition and proper synaptic function, appears to interact with all three PDZ domains of PSD-95 [75] (Fig. 1). As previously reported, PSD-95 organizes a cytoskeletal signaling complex at the postsynaptic membrane. The trafficking of this complex to the postsynaptic sites depends on specific motor proteins as myosin-V and cytoplasmic dynein, which both share the dynein light chain (DLC) that colocalizes with PSD-95 in dendritic spines [76]. Moreover, the accepted and well-known functions of PSD-95 include regulating the maturation of the dendritic spines [17], stabilizing dendrite branching and outgrowth [77], inducing LTP [78, 79], restructuring the presynaptic terminal [80], and modulating the trafficking and synaptic localization of NR2A-rich and NR2B-rich receptors during development [81]. It has been reported that PSD-95 plays a fundamental role in dendritic branching during the development of immature neurons; in fact, the developmental increase in the synaptic expression of PSD-95 was shown to block the synaptic clustering of NR2B-NMDARs, impeding reactivation of synaptic branching [82]. By contrast, PSD-95 has been shown to be an essential component during the maturation of postmitotic neurons because its gene transcription is regulated antagonistically by two enzymes that alter chromatin structure via specific epigenetic modifications [83]. Appropriate levels of PSD-95 are specially required for activity-dependent synapse stabilization after the initial phases of LTP; this process depends on the incorporation of AMPARs at the synapse [84]. However, in vivo studies have revealed that during the development of the visual cortex after eye opening, PSD-95 is rapidly transported from the soma to the synapse [85]. These results suggest, as shown below, that PSD-95 is not static at the synapse but is transported dynamically inside and outside the postsynaptic membrane, a mechanism that is regulated by synaptic activity. In this context, it has been reported

that NMDARs activate the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway, which acts through brainderived neurotrophic factor (BDNF) to induce the intracellular transport of PSD-95 to the postsynaptic membrane [86]. Moreover, it has been reported that in cultured hippocampal cells, PSD-95 is mobilized to the synaptic region of dendritic spines through the activation of the Wnt signaling pathway [22].

Posttranslational Modifications That Regulate the Synaptic Localization of PSD-95 The synaptic localization of PSD-95 can be regulated through various posttranslational modifications depending on developmental stage, synaptic activity, and disease. The residues, enzymes, and mechanisms involved in some of these modifications, including phosphorylation, are well understood; however, for other processes, several pieces of the puzzle are missing. In this section, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 (Tables 1 and 2; Figs. 2 and 3), describing their functional consequences and the signaling pathways that induce such changes. Palmitoylation Palmitoylation is the process by which palmitic acid, a saturated 16-carbon fatty acid, is added to specific cysteine residues via the formation of a thioester bond (thiopalmitoylation or S-palmitoylation). Lipid modifications increase the hydrophobicity of proteins, affecting both their structure and their affinity for cellular membranes or for specific domains within the cell membrane [87, 88]. Palmitoylation, in contrast to other stable lipid modifications such as myristoylation and prenylation, is a labile and reversible posttranslational modification that allows the dynamic regulation of protein targeting [89]. Previous studies have indicated that the palmitoylation of PSD-95 at two conserved cysteine residues (C3 and C5) located in the N-terminal region [90] (Fig. 2) is necessary for sorting to dendrites, participation in synaptic transmission, and the formation of PSD-95 clusters at the postsynaptic membrane of the PSD [91]. Similarly, numerous classes of neuronal proteins containing cysteine residues are susceptible to palmitoylation, including neurotransmitter receptors, synaptic scaffolding proteins, and secreted signaling molecules. The first neuronal protein found to contain covalently attached palmitate was rhodopsin [92]. Additionally, neuronal growth-associated protein 43 (GAP43) [93], most α-subunits of G proteins [94], and the neural cell adhesion molecule NCAM140 [95], among others, are regulated by palmitoylation [96].

Mol Neurobiol Table 1

Posttranslational modifications of PSD-95 and their role in synaptic function

Posttranslational modification

Effector agent

Location

Palmitoylation

DHHC2 Cys3, Cys5

S-nitrosylation

NO

Cys3, Cys5

Ubiquitination Neddylation

Mdm2 Nedd8

Lys10, Lys403, Lys544, Lys672, Lys679 Lys202

Function

Reference

Facilitation of the entry of PSD-95 into the postsynaptic site and its stabilization within the PSD Synaptic transmission Regulation of PSD-95 targeting to synapses blocking free cysteines andmaintaining PSD-95 in the depalmitoylated state Regulation of glutamatergic synapses, synaptic strength, and plasticity Promotion of spine stability, PSD-95 clustering, and spine maturation

[1–3]

[4] [5–7] [8]

The table summarizes different posttranslational modifications involved in the postsynaptic localization of PSD-95. Here, several proteins that act in specific residues of PSD-95 are shown together with their correspondent synaptic alterations

Palmitoylation levels are determined by specific palmitoyl acyl transferases (PATs) and palmitoyl protein thioesterases (PPTs). The DHHC (Asp-His-His-Cys) protein family has been characterized as a group of palmitoylating enzymes with distinctive subcellular localizations. In fact, the DHHC3 and DHHC2 subfamilies are located in the Golgi complex and the dendritic spine, respectively; these proteins are PATs for PSD-95 in neurons [97, 98]. Moreover, it has been reported that although both DHHC3 and DHHC2 are required for the accumulation of PSD-95 at the postsynaptic site, remarkably, only DHHC2 is needed for the dynamic palmitoylation of PSD-95 induced by reduced synaptic activity [99] (Table 1). Once PSD-95 reaches its destination at the postsynaptic region, DHHC2 maintains the equilibrium between palmitoylated PSD-95 and nonpalmitoylated cytosolic PSD-95 in the dendritic shafts. A further comprehension of the possible biological functions of the palmitoylation of PSD-95 requires an understanding of the mechanism by which it occurs. Palmitate turnover Table 2

on PSD-95 is dynamically regulated by glutamate receptor activity. Glutamate-induced synaptic activity triggers the depalmitoylation of PSD-95, possibly mediated by a depalmitoylating enzyme, PPT, which disperses synaptic clusters of PSD-95 and consequently triggers a selective loss of synaptic AMPARs through lateral diffusion within the membrane. This causes the dissociation of PSD-95 from synaptic sites and the endocytosis of AMPARs [100] (Table 1). Conversely, a blockade of activity via tetrodotoxin or treatment with a glutamate receptor antagonist causes an increase in palmitoylated PSD-95 and leads to its accumulation at synaptic sites due to the translocation of DHHC2 [99]. Subsequently, synaptic AMPARs are recruited to defined sites, suggesting that the palmitoylationdepalmitoylation cycle of PSD-95 contributes bidirectionally to AMPAR homeostasis (Fig. 4). The precise mechanism by which PSD-95 is palmitoylated and depalmitoylated is not well documented, and the specific PPT has not yet been identified. Several studies have described numerous approaches

Phosphorylations of PSD-95 and their role in physiological synaptic function

Effector agent

Location

Function

Reference

c-Abl kinase

Tyr533

[9]

SrcPTKs CDK5 CK2

Tyr523 Tyr19, Ser25, Ser35 Thr/Ser

JNK1

Ser295

SAPK3

Thr287, Ser290

GSK-3β

Thr19

CaMKII

Ser73

Modulation of synapse formation by mediating PSD-95 clustering at postsynaptic sites Upregulation of NMDAR function and synaptic transmission Regulation of PSD-95/NMDAR clustering at synapses Regulation of the interaction of NMDARs with PSD-95 as well as surface NMDAR expression Synaptic accumulation of PSD-95 triggering synaptic potentiation through the recruitment of AMPARs Regulation of protein-protein interactions at the synapses in response to adverse stress- or mitogen-related stimuli Destabilization of PSD-95 within the PSD impairing AMPARs internalization and the induction of LTD Regulation of the signaling transduction pathway downstream of NMDARs and modulation of the spine growth and synaptic plasticity

[10, 11] [12] [13, 14] [15, 16] [17] [18] [19–21]

The table summarizes the role of different kinases that phosphorylate PSD-95 at specific residues and are involved in its postsynaptic localization

Mol Neurobiol

Fig. 2 Posttranslational modifications of specific PSD-95 residues regulate its synaptic localization. The diagram illustrates the schematic structure of PSD-95 composed of the independent modular domains PDZ1, PDZ2, PDZ3, SH3, and GK. The figure shows the corresponding localized residues at which different posttranslational

modifications can occur to alter the synaptic localization of PSD-95. The upper panel depicts the palmitoylation, S-nitrosylation (green spectrum), and phosphorylation (purple) processes and their specific residues. The lower panel shows the ubiquitination (blue) and neddylation (red) processes and their specific residues

aimed at determining the roles of PSD-95 palmitoylation in synaptic transmission, PSD organization, and synaptic

plasticity. A recent study has provided new data about the role of local palmitoylation machinery within the

Fig. 3 The synaptic fate of PSD-95 is defined through the action of different posttranslational modifications. The scheme shows a central synapse. At the dendritic spine, different posttranslational modifications such as palmitoylation, S-nitrosylation, phosphorylation, ubiquitination, and neddylation control the postsynaptic localization of PSD-95. Shown are the most representative residues where these modifications act. The

right side shows the mechanisms that trigger the accumulation of PSD-95 and the subsequent increase in its clustering within the PSD. These mechanisms enhance synaptic transmission. The left side shows the posttranslational modifications that destabilize PSD-95 within the PSD and, in some cases, lead to the translocation of the protein out of the active spine, thereby disrupting spine growth and synaptic plasticity

Mol Neurobiol

Fig. 4 Regulation of the palmitate turnover on PSD-95 within the dendritic spine. The figure shows the postsynaptic region of a synapse. Increased glutamate receptor activity causes the depalmitoylation of PSD95, possibly via a putative PPT, triggering a decrease in PSD-95 clustering at synaptic sites, the lateral diffusion of AMPARs, and their

consequent endocytosis (right panel). By contrast, an activity blockade leads to the translocation of DHHC2 from the dendritic shaft to the synapse, palmitoylating PSD-95 and therefore increasing its clustering and inducing the recruitment of synaptic AMPARs (left panel)

dendritic spines. It has been discovered that palmitoylated PSD-95 molecules by DHHC2 are grouped in subsynaptic nanodomains that are essential for the maintenance of the organization of the PSD-95 clusters in the PSD [101]. Three studies published in 2002 associated the dynamic palmitate cycling of PSD-95 with synaptic plasticity because palmitoylated PSD-95 interacts with the AMPAR trafficking protein Stargazin. Stargazin is known to target PSD-95 complexes to the plasma membrane, thus confirming that the palmitoylation of PSD95 is essential for the modulation of synaptic AMPARs [49, 100, 102]. Otherwise, distinct domains of PSD-95, specifically PDZ1 and PDZ2, contribute to securing PSD-95 within the PSD, whereas the C-terminal SH3 domain is involved in the formation of a stable lattice of PSD-95 molecules that stabilizes PSD-95 in the dendritic spine. The N-palmitoylation of PSD-95 and the protein interaction mediated by both PDZ domains are necessary for facilitating the entry of PSD-95 into the postsynaptic region [9]. Regarding the role of NMDARs in synaptic transmission, it is thought that the palmitoylation of PSD-95 affects NMDAR synaptic trafficking, clustering, and

function as, conversely, the activation of NMDAR destabilizes PSD-95, releasing PSD-95 molecules from the dendritic spine [9]. Several studies have confirmed this assumption; in fact, palmitoylated PSD-95 alters NMDAR destabilization, affecting NMDAR activation during prolonged exposure to glutamate [103]. Moreover, it was found that NMDAR clustering was not altered in the presence of 2-bromopalmitate (2-BP), a palmitoylation inhibitor. This may be due to the direct effects of this inhibitor or to the accumulation of nonpalmitoylated PSD-95 [80, 100]. Furthermore, the palmitoylation of PSD-95 may be related to the number and size of dendritic spines because enhanced synaptic clustering of PSD-95 is required for its palmitoylation [80]. Thus, PSD-95 palmitoylation may play an important role in maintaining synaptic function, and therefore, changes in the synaptic stability of PSD-95 may be involved in the control of postsynaptic maturation and synaptic transmission. Although many studies have explored the association of PSD-95 palmitoylation with glutamate receptor function related to synaptic distribution, other roles involving the regulation of synaptic plasticity have been described. BDNF and tropomyosin receptor kinase B

Mol Neurobiol

(TrkB) signaling activation are necessary for PSD-95 palmitoylation mediated by the palmitoylating enzyme ZDHHC8 [104]. Moreover, recent novel studies have established that the postsynaptic localization of PSD-95 could be regulated through its palmitoylation and mediated by various downstream pathways, molecules, and enzymes [105–107]. In particular, it has been demonstrated that Ca 2+ /calmodulin (CaM) binds at the Nterminus of PSD-95, blocking palmitoylation, which promotes Ca 2+ -induced dissociation of PSD-95 from the postsynaptic membrane [107]. S-nitrosylation Reactive nitrogen species, such as nitric oxide (NO), exert their biological effects through posttranslational modifications of cysteines, forming S-nitrosothiols [108]. This chemical reaction is part of a process termed protein S-nitrosylation, which affects protein function similar to phosphorylation and plays a crucial role in the pathogenesis of neurodegenerative diseases [109, 110]. Nitrosylation is considered to be one of the major posttranslational modifications of proteins. Genetic evidence indicates that this protein modification reaction has diverse regulatory roles [111, 112]. PSD-95 is physiologically S-nitrosylated at cysteines 3 and 5 (C3 and C5) by NO, and this S-nitrosylation has a reciprocal relationship with palmitoylation (Fig. 2) that affects the physiologic clustering of PSD-95 at synapses and impacts its principal functions [113] (Table 1). As previously indicated, the dynamic cycling of palmitoylation and depalmitoylation regulates the function of PSD-95. The activation of the glutamate receptor increases the depalmitoylation of PSD-95 [100]. Calcium enters the cell via NMDA ion channels and binds to calmodulin associated with nNOS, causing NO formation. This NO S-nitrosylates PSD-95 at both C3 and C5 in a process that competes with palmitoylation, blocking free cysteines and maintaining PSD-95 in the depalmitoylated form [113]. Although some PPTs have been described, including APT1 and PPT1 [96], none of them has been shown to act directly upon PSD-95. Thus, NO may act in conjunction with these depalmitoylating enzymes, preventing the palmitoylation of PSD-95 and thus maintaining higher levels of depalmitoylated PSD-95. Conversely, endogenous palmitoylation reduces the levels of S-nitrosylated PSD-95. Phosphorylation Numerous protein kinases are involved in the phosphorylation of serine, threonine, and tyrosine residues, r e s u l t i n g i n m o d i f i c a t i o ns o f p r o t e i n f u n c t i o n [114–116]. Reversible protein phosphorylation has been

known to control a wide range of biological functions and activities [117]. Various models of synaptic activation that regulate kinases and/or phosphatases and simultaneously target postsynaptic proteins have been described. It is now well accepted that postsynaptic protein phosphorylation/ dephosphorylation events regulate the trafficking of receptors to and from the synaptic region and are essential steps in activity-induced modification of synaptic efficacy [118–123]. Recent evidence suggests that the trafficking and turnover of scaffolding molecules that determine the organization of the PSD are regulated by phosphorylation/ dephosphorylation events. Various studies have demonstrated that PSD-95 is phosphorylated at specific residues (Fig. 2) by several kinases that posttranslationally modulate proper clustering of this protein (Table 2). The kinase c-Abl is primarily present in the postsynaptic compartment, in which it phosphorylates PSD-95 on tyrosine 533. The inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to a decrease in PSD-95 clustering at postsynaptic sites and a consequent decrease in the number of synapses [124]. Other kinases have also been reported to phosphorylate PSD-95. Some of the Src family protein tyrosine kinases (SrcPTKs) can phosphorylate PSD-95 on tyrosine 523 after brain ischemia, altering the activation of NMDARs [125]. Moreover, it has been demonstrated that this PSD-95 phosphorylation facilitates the subsequent tyrosine phosphorylation of NR2A, resulting in the upregulation of NMDAR function and synaptic transmission [126]. Another group of phosphorylation sites in the N-terminal domain of PSD-95 (tyrosine 19, serine 25, and serine 35) has been described as target sites for cyclin-dependent kinase 5 (CDK5), and activated CDK5 reduces the ability of PSD-95 to multimerize and to augment the size of its clusters [127]. Phosphorylation of different serine/threonine sites on PSD-95 may trigger dynamic posttranslational regulation of PSD-95/NMDAR clustering at synapses. Regarding modifications that affect NMDARs, it has been reported that casein kinase II (CK2) phosphorylates the serine 1480 residue of the PDZ NR2B subunit. This phosphorylation regulates the interaction of NMDARs with PSD95 and modulates the function and plasticity of excitatory synapses [128]. In fact, one study evaluated the effects of CK2 activity on NMDARs in the presence of a selective CK2 inhibitor and reported that PSD-95 was a CK2 substrate that uses GTP as a phosphoryl donor. Thus, that report confirmed the results of previous studies and demonstrated that NMDARs are directly regulated by CK2 and indirectly regulated by PSD-95 [129]. Phosphorylation at serine 295, which is located between the PDZ2 and PDZ3 domains of PSD-95,

Mol Neurobiol

regulates its postsynaptic localization, as well as the synaptic localization of the aforementioned molecules that are bound to these second and third PDZ domains [130]. However, synaptic accumulation of PSD-95 is essential for potentiating synaptic transmission, and it has been reported that this serine 295 phosphorylation, mediated by JNK1 and dependent on Rac1, promotes synaptic accumulation of PSD-95 and influences synaptic potentiation, thus affecting LTD. In addition, dephosphorylation of serine 295 by the phosphatases PP1 and PP2A is known to be required for AMPAR internalization and LTD [131]. These data are consistent with several studies indicating that the postsynaptic abundance of PSD-95 establishes synaptic strength by controlling AMPAR trafficking [49, 50, 79, 80]. Several functions of PSD-95 have been reported to be regulated by phosphorylation (Table 2). The regulation of protein-protein interactions at the synapses could be modulated by the phosphorylation of PSD-95 at threonine 287 and serine 290 by SAPK3/p38γ in response to stress- or mitogen-related stimuli [132]. PSD-95 phosphorylation at threonine 19 by glycogen synthase kinase-3β (GSK-3β) triggers PSD-95 destabilization within the PSD and is a critical step for AMPAR mobilization and LTD [133]. It is also known that the phosphorylation of PSD-95 at serine 73, located in the PDZ1 domain, by CaMKII is responsible for dynamic regulation of the signal transduction pathway downstream of NMDARs [68, 134]; moreover, this phosphorylation destabilizes PSD-95 in the PSD, triggering activity-dependent trafficking of PSD-95 out of the active dendritic spine and disrupting spine growth and synaptic plasticity [135]. Ubiquitination The PDZ domain-containing proteins are regulated by the ubiquitin pathway [136], and many aspects of synaptic function, including the regulation of PSD-95, seem to be controlled by the ubiquitin-proteasome pathway. The ubiquitination of presynaptic proteins is known to negatively regulate synaptogenesis, thereby modifying the efficacy of synaptic transmission [137]. In fact, the attachment of ubiquitin performs a crucial role in the regulation of synaptic function and neurotransmitter release as well as in the alteration of PSD and plasticity, spine growth, and stability [138–140]. Ubiquitin is a 76-amino-acid globular protein that is covalently attached to substrate proteins. Once tagged with ubiquitin [141], substrate proteins are often targeted for rapid degradation by the 26S proteasome [142]. The ubiquitin-activating (E1), ubiquitinconjugating (E2), and ubiquitin-ligating (E3) enzymes

are key regulators of protein ubiquitination [141, 143]. Protein ubiquitination begins with the formation of a thiol-ester linkage between the C-terminus of ubiquitin and the active site cysteine of E1 [144, 145]. Therefore, ubiquitinated proteins are regulated at three levels: first, by modification of the substrate; second, by modulation of the activity of ubiquitin ligases; and third, by the removal of ubiquitins [138, 146]. The ubiquitination of PSD-95, which is mediated by the ubiquitin E3 ligase murine double minute 2 (Mdm2), results in the subsequent removal of the scaffold protein from synaptic sites by proteasome-dependent degradation in response to NMDAR activation, with the consequent loss of PSD-95 clustering [147]. Loss of PSD-95 presumably untethers AMPARs from the postsynaptic membrane, allowing for their subsequent removal from synaptic sites by endocytosis. Thus, the ubiquitin-proteasome system might regulate the molecular architecture of glutamatergic synapses and play a fundamental role in synaptic strength and plasticity in the mammalian brain [148]. However, a recent study used a novel approach to indicate that increased interaction between Mdm2 and PSD-95 caused by a reduction in CDK5 enhances PSD-95 ubiquitination without affecting PSD-95 protein levels, thus suggesting a nonproteolytic function for ubiquitination at synapses. Moreover, in this work, the authors identified five lysine residues in PSD-95 that were ubiquitinated (Fig. 2): lysine 10 in the N-terminus; lysine 403 in the linker between the PDZ3 and SH3 domains; and lysines 544, 672, and 679 in the GK domain [149] (Table 1). A recent study suggested that copper may act as a cofactor of the E1-E2-E3 enzymes, promoting the ubiquitination of PSD-95 and the subsequent saturation of the proteasome. It has been reported that under chronic copper release, degradation of PSD-95 slows, leading to AMPAR clustering at the plasma membrane and enhancement of synaptic neurotransmission [150]. Other recent studies have focused on brain-specific angiogenesis inhibitor 1 (BAI1). Although BAI1 has been mainly studied as a negative regulator of angiogenesis and tumorigenesis [151, 152], some evidence has indicated that it is highly expressed in neurons [153]. A novel function of BAI1 as a critical regulator of synaptic plasticity at hippocampal excitatory synapses in the brain has recently been revealed: BAI1 interacts with Mdm2 to prevent PSD-95 polyubiquitination and degradation. Therefore, the association of BAI1 with Mdm2 regulates synaptic plasticity and stabilizes PSD-95 [154]. Neddylation Neddylation is a posttranslational protein modification resembling ubiquitination, in which neural precursor cell-expressed developmentally downregulated gene 8

Mol Neurobiol

(Nedd8) is conjugated to its target substrates via a substrate-specific ligase. Neddylation has a particular activating enzyme (Nedd8-activating enzyme, NAE) and a conjugating enzyme (Ubc12) [155, 156]. The mRNAs encoding Nedd8 and Ubc12 are highly expressed in hippocampal neurons, suggesting that neddylation is important for synaptic plasticity and controls spine development during neuronal maturation and spine stability in mature neurons. Furthermore, neddylation specifically influences the development of excitatory synapses, increases the number of synapses, and is involved in the maintenance of dendritic spines [157] (Table 1). One of the targets mediating the effect of neddylation on dendritic spines is PSD-95. In fact, PSD-95 at synapses is endogenously neddylated at lysine 202 (Fig. 2), which is located in the second PDZ domain. This is the first evidence that a synaptic protein (PSD-95) can act as a substrate of Nedd8, and it illustrates the synaptic functions of neddylation in mature neurons (i.e., the promotion of spine stability). Moreover, neddylation has been implicated in the scaffolding function of PSD-95, as it prevents PSD-95 declustering and consequent diffusion out of the dendritic spine. Regarding the role of PSD-95 in the maturation of spines and excitatory synapses, it has been shown that neddylation of lysine 202 is essential for PSD-95 function. Several lines of evidence indicate that PSD-95 is present in nascent spines and plays a role in spine maturation in neuronal cultures [80, 157]. Furthermore, it has recently been reported that in addition to its ability to ubiquitinate PSD-95 [148], the ubiquitin ligase Mdm2 mediates the neddylation of PSD-95 [158]; however, neddylation does not affect the expression level of PSD-95, and the possible cross talk between ubiquitination and neddylation remains unknown [158].

Regulation of PSD-95 Function and Posttranslational Modifications Involved in Neurological Diseases Posttranslational modifications of PSD-95 regulate several aspects of its function and, by extension, modulate the fate of excitatory synaptic transmission. Therefore, it is not surprising that the alteration of one or more pathways controlling such modifications can lead to the development of neurological diseases. One remarkable example is Alzheimer disease (AD), which has been associated with several alterations in PSD-95. It is known that in brain aging, particularly during the early stages of AD, amyloid-β (Aβ) oligomers can bind preferentially to postsynaptic regions and

may bind directly to NMDARs and to NL-1 [159, 160]. Specifically, it has been suggested that the interaction between NL-1 with Aβ, which increases the formation of Aβ oligomers, could induce the targeting of Aβ oligomers to the postsynaptic region of excitatory synapses [161]. As we have mentioned before, NMDARs and NL-1 interact directly with PSD-95. It has even been suggested that Aβ may interact directly with PSD-95, causing synaptic damage under certain pathological conditions; in fact, some reports have shown coimmunoprecipitation of Aβ with PSD-95. In addition, it has been observed that Aβ co-localizes with PSD-95 specifically at excitatory synapses in human postmortem AD brains as well as in cultured murine neurons exposed to Aβ oligomers [162, 163]. Several reports have demonstrated decreased levels of PSD-95 in pathologically vulnerable regions of the brain in AD patients [164]. Part of the mechanism responsible for this decrease involves the ubiquitin-proteasomal degradation of PSD-95. This was confirmed in SK-N-MC cells transfected with a mutant version of PSD-95 that lacks the PEST (proline (P), glutamic acid (E), serine (S), and threonine (T)-rich) sequence, which is essential for its ubiquitination [148]. Treatment of these cells with Aβ failed to induce the decrease in PSD-95 observed when the cells were transfected with the WT form of PSD-95 [165]. This result is consistent with the fact that dysfunction of the ubiquitination or deubiquitination machinery, mutations in ubiquitin, proteasomal impairment, and mutations in proteasome substrates affecting their rate of degradation underlie the pathogenesis of many neurodegenerative diseases. PSD-95 modulation is a key step in the pathological progression induced by Aβ oligomers. The loss of PSD95 at synapses inevitably results in PSD disassembly, as evidenced by the concomitant loss of other synaptic partners such as NMDARs [166], AMPARs [167], and SynGAP [168]. Interestingly, treatment with Wnt-5a, a synaptogenic ligand that signals through a β-cateninindependent pathway, decreases Aβ-induced synaptic damage, protecting cultured hippocampal neurons from dendritic spine loss and from decreased synaptic expression of PSD-95, SynGAP, and glutamate receptors [21, 22, 168, 169] (Fig. 5). Therefore, molecules and signaling pathways that regulate the integrity and function of PSD-95 may have therapeutic potential for reducing Aβ-induced synaptic loss and cognitive impairment in AD. Another hypothesis related to the pathogenic progression of AD also involves PSD-95. In AD models, the ability of PSD-95 to interact with other synaptic partners is impaired; for example, tau protein, which is normally located in axons, migrates to dendrites. This allows the translocation of the Src kinase Fyn, which

Mol Neurobiol

Fig. 5 Wnt-5a prevents the Aβ oligomer-induced changes in PSD-95 clustering in hippocampal neurons. a Representative images of neurite immunofluorescence labeling of PSD-95 (green) and fluorescent labeling of processes with phalloidin (blue) in neurons incubated in the control medium (a), medium containing Wnt-5a (b), and medium containing Wnt-5a plus soluble Frizzled receptor protein-1 (sFRP) (c) after 1 h of treatment. b Time course of the effect of Wnt-5a on PSD-95, as analyzed

by quantification of PSD-95 clusters/100 μm of neurite (n = 3) (modified from Farías et al. [22]). c Representative images of neurite double immunofluorescence labeling of PSD-95 (green) and synapsin-1 (red) and phalloidin staining (blue) in samples subjected to control, Aβ, and Aβ/Wnt-5a treatments for 1 h. Merged images show the apposition of the presynaptic (red) and postsynaptic (green) boutons. d Quantification of synaptic PSD-95 over total PSD-95 (modified from Cerpa et al. [169])

phosphorylates the Y1472 residue of the NR2 subunit, to facilitate interaction of the NMDAR complex with PSD-95, linking NMDARs to synaptic excitotoxic downstream signaling. This process suggests that formation of stable NMDAR/PSD-95 complexes is required for Aβ toxicity [170] (Fig. 6a). There are several neurological disorders in which impairments in the normal function of PSD-95 are associated with posttranslational modifications. Regarding palmitoylation, different PATs have been linked to AD, schizophrenia, Huntington disease, X-linked mental retardation, and infantile- and adult-onset forms of neural ceroid lipofuscinosis in humans [171]. For example, ZDHHC9 [172] and ZDHHC15 [173] have been associated to a subtype of X-linked mental retardation (XLMR) in humans, and ZDHHC12 has been linked to the regulation of amyloid precursor protein (APP) trafficking and Aβ generation [174]. However, to date, only three PATs have been linked to neuropathologies in which PSD-95 is involved. A single-nucleotide polymorphism (SNP) associated with schizophrenia is located in the DHHC8 gene [175–177], and a significant

reduction in PSD-95 palmitoylation has been observed in the presence of that SNP (Fig. 6b). Huntington disease, which is caused by alterations in the structure of huntingtin protein (HTT), has been directly linked to DHHC17 (also known as huntingtin interacting protein 14, HIP14) because, in addition to palmitoylating PSD-95 [178], DHHC17 regulates the trafficking and function of HTT through its palmitoylation, modulating the formation of inclusion bodies and neuronal toxicity [179]. Moreover, HTT with 18 glutamine repeats (YAC18, nonpathogenic) can palmitoylate PSD-95 and is involved in its synaptic trafficking [105] (Table 3) (Fig. 6c). By contrast, activation of the myocyte enhancer factor 2 (MEF2) family together with the fragile X mental retardation protein (FMRP), an RNA binding protein, is necessary in synapse elimination [180, 181], a process that is ultimately mediated by the ubiquitination of PSD-95 by Mdm2. However, the gene that encodes FMRP is transcriptionally silenced in patients with fragile X syndrome (FXS), the most common inherited form of human cognitive dysfunction and autism [182].

Mol Neurobiol

Fig. 6 Neurological diseases associated with posttranslational modifications of PSD-95. The figure presents the postsynaptic region of a synapse in four neurological disease contexts in which PSD-95 is posttranslationally modified and, by extension, its normal function is disrupted. a In AD neuropathology, the accumulation of Aβ oligomers occurs preferentially in the postsynaptic region, triggering various scenarios, including the ubiquitin-proteasomal degradation of PSD-95. Consequently, the PSD is disassembled, causing the loss of NMDARs and SynGAP. On the other hand, tau protein migrates to dendrites, allowing the translocation of Fyn, which results in the phosphorylation of the NR2 subunit and thus in excitotoxicity. Both situations result in the loss of synapses. b In patients with schizophrenia, several microdeletions can be observed in the DHHC8 gene, where the relevant SNP is located. Therefore, in a pathological context, the palmitoylation of PSD-95 is reduced and a decreased density of dendritic spines and glutamatergic synapses is observed (right spine). In a healthy context, the

palmitoylating enzyme DHHC8 is expressed, and it exerts its normal function of palmitoylating PSD-95 (left spine). c In Huntington disease, the structure of HTT is altered, and DHHC17, which is directly related to HTT, is unable to palmitoylate either HTT or PSD-95. Consequently, the clustering of PSD-95 is reduced (right spine). In the absence of disease, DHHC17 palmitoylates both HTT and PSD-95. Moreover, YAC18, which is nonpathogenic, is also able to palmitoylate PSD-95 (left spine). d In the absence of disease, two main mechanisms are involved in the ubiquitination of PSD-95, a necessary process for synapse elimination, in which MEF2 and FMRP are key components. On the one hand, Mdm2 mediates the ubiquitin-proteasomal degradation of PSD-95. In addition, Pcdh10 also contributes to the ubiquitination of PSD-95, delivering the ubiquitinated PSD-95 to the proteasome (left spine). In patients with FXS, FMRP is not expressed, resulting in a decrease in PSD-95 degradation and an excessive number of dendritic spines (right spine)

Moreover, protocadherin 10 (Pcdh10) is required for MEF2-induced synapse elimination because it acts by delivering ubiquitinated PSD-95 to the proteasome; therefore, the defective degradation of PSD-95 may explain the excessive number of dendritic spines observed in FXS [183] (Table 3) (Fig. 6d). In addition, a recent study has demonstrated that FMRP co-localizes with PSD-95 [184]; it is therefore plausible that the scaffold

protein PSD-95 may be associated with the pathobiology of FXS. After describing the broad spectrum of interactions permitted by the conformationally independent modular PDZ domains and reviewing several neuropathologies associated with the posttranslational modifications of PSD-95, it is worth mentioning that various studies have reported that PSD-95 is a potential therapeutic

Mol Neurobiol Table 3

Posttranslational modifications of PSD-95 involved in different neuropsychiatric diseases and their functional and pathological consequences

Posttranslational modification

Neuropathology

Effector agent

Effects

Reference

Palmitoylation

Schizophrenia

ZDHHC8

[22]

Palmitoylation

Huntington’s disease

DHHC17/HIT14

Palmitoylation

Huntington’s disease

YAC18

Ubiquitination

FXS

Mdm2

Reduction in PSD-95 palmitoylation associated with a synaptic loss Reduction in PSD-95 palmitoylation associated with a synaptic loss Increase in PSD-95 palmitoylation and augmentation in PSD-95 clustering at synaptic sites Increase in PSD-95 clustering and excessive dendritic spine number

[23] [24]

[25]

The table summarizes different posttranslational modifications that are associated with neurological disorders. Here, several proteins are shown together with the effect that cause in PSD-95

target for a wide range of diseases. The design of dimeric [185] and trimeric [186] ligands of PSD-95, the synthesis of peptides that interact with its PDZ domains [45, 187–189], and the employment of brain-specific proteins including calcyon [190], which forms a complex with PSD-95 that links glutamatergic and dopaminergic signaling, make PSD-95 a potential therapeutic target for the modulation of numerous neuropsychiatric disorders.

of PSD-95 is reduced and its mobility is increased, which may reflect changes in its ubiquitination and palmitoylation status. Thus, although several recent publications have addressed this topic, novel pharmacological studies aimed at inhibiting the posttranslational modification of PSD-95 are necessary. Finally, signaling pathways upstream of the posttranslational modification of PSD-95 have also been shown to regulate various aspects of synaptic physiology, and mutation studies with PSD-95 have demonstrated that this regulation is necessary for proper synaptic development and plasticity.

Conclusions

Acknowledgments This work was supported by grants from the Basal Center of Excellence in Aging and Regeneration (CONICYT-PFB 12/ 2007) and FONDECYT (No. 1120156) to N. C. Inestrosa. D. Vallejo and J. F. Codocedo were postdoctoral fellows of CARE. We also thank the Sociedad Química y Minera de Chile (SQM) for a special grant on “The Effects of Lithium on Health and Disease”.

PSD-95 has been the best-studied scaffold protein of the PSD since its identification in the early 1990s. A widely accepted function of PSD-95 and its family members is the binding/ tethering or stabilization of various membrane proteins and signaling molecules within the PSD in excitatory chemical synapses. This protein is therefore centrally involved in multiple aspects of synaptic function. Consistent with this idea, altering the abundance of PSD-95 scaffolds at synapses is a primary means of controlling PSD-95 function and synaptic strength. In fact, as we have reviewed here, synaptic activity leads to depalmitoylation of PSD-95, which is correlated with the depletion of synaptic PSD-95 clusters, loss of AMPARs, and weakening of synapses [102]. Activity-induced ubiquitination of PSD-95 and the subsequent loss of PSD-95 from synapses via proteasomal degradation are also implicated in synaptic depression [148]. Chronic changes in the abundance of postsynaptic PSD-95 regulated by ser-295 phosphorylation may contribute to homeostatic plasticity (synaptic scaling) [131]. Nitrosylation and the recently described neddylation of PSD-95 also contribute to its synaptic localization and synaptic stability. Considering that the maintenance of a population of stable synaptic connections is probably critically important for the preservation of memory and functional circuitry, understanding the molecular dynamics underlying synapse stabilization is essential. In fact, in several neuropathologies, such as AD and Huntington disease, the level

References 1. 2.

3.

4.

5. 6.

7.

Kim E, Sheng M (2009) The postsynaptic density. Curr Biol. doi: 10.1016/j.cub.2009.07.047 DeGiorgis JA, Galbraith JA, Dosemeci A, Dosemeci A et al (2006) Distribution of the scaffolding proteins PSD-95, PSD-93, and SAP97 in isolated PSDs. Brain Cell Biol 35:239–250. doi:10. 1007/s11068-007-9017-0 Chen X, Vinade L, Leapman RD et al (2005) Mass of the postsynaptic density and enumeration of three key molecules. Proc Natl Acad Sci U S A 102:11551–11556. doi:10.1073/pnas. 0505359102 Feng W, Zhang M (2009) Organization and dynamics of PDZdomain-related supramodules in the postsynaptic density. Nat Rev Neurosci 10:87–99. doi:10.1038/nrn2540 Kim E, Sheng M (2004) PDZ domain proteins of synapses. Nat Rev Neurosci 5:771–781. doi:10.1038/nrn1517 Blanpied TA, Kerr JM, Ehlers MD (2008) Structural plasticity with preserved topology in the postsynaptic protein network. Proc Natl Acad Sci U S A 105:12587–12592. doi:10.1073/pnas. 0711669105 Kerr JM, Blanpied TA (2012) Subsynaptic AMPA receptor distribution is acutely regulated by actin-driven reorganization of the postsynaptic density. J Neurosci 32:658–673. doi:10.1523/ JNEUROSCI.2927-11.2012

Mol Neurobiol 8.

Kuriu T, Inoue A, Bito H et al (2006) Differential control of postsynaptic density scaffolds via actin-dependent and -independent mechanisms. J Neurosci 26:7693–7706. doi:10.1523/ JNEUROSCI.0522-06.2006 9. Sturgill JF, Steiner P, Czervionke BL, Sabatini BL (2009) Distinct domains within PSD-95 mediate synaptic incorporation, stabilization, and activity-dependent trafficking. J Neurosci 29:12845– 12854. doi:10.1523/JNEUROSCI.1841-09.2009 10. Sheng M, Kim E (2011) The postsynaptic organization of synapses. Cold Spring Harb Perspect Biol. doi:10.1101/cshperspect. a005678 11. Chen X, Nelson CD, Li X et al (2011) PSD-95 is required to sustain the molecular organization of the postsynaptic density. J Neurosci 31:6329–6338. doi:10.1523/JNEUROSCI.5968-10. 2011.PSD-95 12. Hunt CA, Schenker LJ, Kennedy MB (1996) PSD-95 is associated with the postsynaptic density and not with the presynaptic membrane at forebrain synapses. J Neurosci 16:1380–1388 13. Irie M, Hata Y, Takeuchi M et al (1997) Binding of neuroligins to PSD-95. Science 277:1511–1515. doi:10.1126/science.277.5331. 1511 14. Kornau HC, Schenker LT, Kennedy MB, Seeburg PH (1995) Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269:1737–1740. doi:10.1126/science.7569905 15. Kim E, Naisbitt S, Hsueh YP et al (1997) GKAP, a novel synaptic protein that interacts with the guanylate kinase-like domain of the PSD-95/SAP90 family of channel clustering molecules. J Cell Biol 136:669–678. doi:10.1083/jcb.136.3.669 16. Tejedor FJ, Bokhari A, Rogero O et al (1997) Essential role for dlg in synaptic clustering of Shaker K+ channels in vivo. J Neurosci 17:152–159 17. El-Husseini AE, Craven SE, Chetkovich DM et al (2000) Dual palmitoylation of PSD-95 mediates its vesiculotubular sorting, postsynaptic targeting, and ion channel clustering. J Cell Biol 148:159–171. doi:10.1083/jcb.148.1.159 18. Migaud M, Charlesworth P, Dempster M et al (1998) Enhanced long-term potentiation and impaired learning in mice with mutant postsynaptic density-95 protein. Nature 396:433–439. doi:10. 1038/24790 19. Inestrosa NC, Varela-Nallar L (2014) Wnt signaling in the nervous system and in Alzheimer’s disease. J Mol Cell Biol 6:64–74. doi: 10.1093/jmcb/mjt051 20. Inestrosa NC, Arenas E (2010) Emerging roles of Wnts in the adult nervous system. Nat Rev Neurosci 11:77–86. doi:10.1038/ nrn2755 21. Varela-Nallar L, Alfaro IE, Serrano FG et al (2010) Wingless-type family member 5A (Wnt-5a) stimulates synaptic differentiation and function of glutamatergic synapses. Proc Natl Acad Sci U S A 107:21164–21169. doi:10.1073/pnas.1010011107 22. Farías GG, Alfaro IE, Cerpa W et al (2009) Wnt-5a/JNK signaling promotes the clustering of PSD-95 in hippocampal neurons. J Biol Chem 284:15857–15866. doi:10.1074/jbc.M808986200 23. Stathakis DG, Hoover KB, You Z, Bryant PJ (1997) Human postsynaptic density-95 (PSD95): location of the gene (DLG4) and possible function in nonneural as well as in neural tissues. Genomics 44:71–82. doi:10.1006/ geno.1997.4848 24. Stathakis DG, Udar N, Sandgren O et al (1999) Genomic organization of human DLG4, the gene encoding postsynaptic density 95. J Neurochem 73:2250–2265. doi:10.1046/j.1471-4159.1999. 0732250.x 25. Bao J, Lin H, Ouyang Y et al (2004) Activity-dependent transcription regulation of PSD-95 by neuregulin-1 and Eos. Nat Neurosci 7:1250–1258. doi:10.1038/nn1342

26.

Wilke SA, Hall BJ, Antonios JK et al (2012) NeuroD2 regulates the development of hippocampal mossy fiber synapses. Neural Dev 7:9. doi:10.1186/PREACCEPT-1243164026616498 27. Guan J-S, Haggarty SJ, Giacometti E et al (2009) HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459:55–60. doi:10.1038/nature07925 28. Muddashetty RS, Nalavadi VC, Gross C et al (2011) Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation, and mGluR signaling. Mol Cell 42:673–688. doi:10.1016/j.molcel.2011.05.006 29. Zheng S, Gray EE, Chawla G et al (2012) PSD-95 is posttranscriptionally repressed during early neural development by PTBP1 and PTBP2. Nat Neurosci 15:381–388. doi:10.1038/nn. 3026 30. Levy JM, Chen X, Reese TS, Nicoll RA (2015) Synaptic consolidation normalizes AMPAR quantal size following MAGUK loss. Neuron 87:534–548. doi:10.1016/j.neuron.2015.07.015 31. Zheng C-Y, Seabold GK, Horak M, Petralia RS (2011) MAGUKs, synaptic development, and synaptic plasticity. Neuroscience 17: 493–512. doi:10.1177/1073858410386384 32. McGee AW, Dakoji SR, Olsen O et al (2001) Structure of the SH3-guanylate kinase module from PSD-95 suggests a mechanism for regulated assembly of MAGUK scaffolding proteins. Mol Cell 8:1291–1301. doi:10.1016/S1097-2765(01)00411-7 33. McCann JJ, Zheng L, Rohrbeck D et al (2012) Supertertiary structure of the synaptic MAGuK scaffold proteins is conserved. Proc Natl Acad Sci 109:15775–15780. doi:10.1073/pnas.1200254109 34. Doyle DA, Lee A, Lewis J et al (1996) Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ. Cell 85:1067– 1076. doi:10.1016/S0092-8674(00)81307-0 35. Craven SE, Bredt DS (2000) Synaptic targeting of the postsynaptic density protein PSD-95 mediated by a tyrosine-based trafficking signal. J Biol Chem 275:20045–20051. doi:10.1074/jbc. M910153199 36. Long JF, Tochio H, Wang P et al (2003) Supramodular structure and synergistic target binding of the N-terminal tandem PDZ domains of PSD-95. J Mol Biol 327:203–214. doi:10.1016/S00222836(03)00113-X 37. Brenman JE, Chao DS, Gee SH et al (1996) Interaction of nitric oxide synthase with the postsynaptic density protein PSD-95 and α1-syntrophin mediated by PDZ domains. Cell 84:757–767. doi: 10.1016/S0092-8674(00)81053-3 38. Hung AY, Sheng M (2002) PDZ domains: structural modules for protein complex assembly. J Biol Chem 277:5699–5702. doi:10. 1074/jbc.R100065200 39. Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes. Annu Rev Neurosci 24:1–29. doi:10. 1146/annurev.neuro.24.1.1 40. Korkin D, Davis FP, Alber F et al (2006) Structural modeling of protein interactions by analogy: application to PSD-95. PLoS Comput Biol 2:1365–1376. doi:10.1371/journal.pcbi.0020153 41. Tompa P (2012) On the supertertiary structure of proteins. Nat Chem Biol 8:597–600. doi:10.1038/nchembio.1009 42. Chen X, Winters C, Azzam R et al (2008) Organization of the core structure of the postsynaptic density. Proc Natl Acad Sci U S A 105:4453–4458. doi:10.1073/pnas.0800897105 43. Niethammer M, Kim E, Sheng M (1996) Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases. J Neurosci 16:2157–2163 44. McCann JJ, Choi UB, Bowen ME (2014) Reconstitution of multivalent PDZ domain binding to the scaffold protein PSD-95 reveals ternary-complex specificity of combinatorial inhibition. Structure 22:1458–1466. doi:10.1016/j.str.2014.08.006

Mol Neurobiol 45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

Cui H, Hayashi A, Sun H-S et al (2007) PDZ protein interactions underlying NMDA receptor-mediated excitotoxicity and neuroprotection by PSD-95 inhibitors. J Neurosci 27:9901–9915. doi: 10.1523/JNEUROSCI.1464-07.2007 Nicoll RA, Tomita S, Bredt DS (2006) Auxiliary subunits assist AMPA-type glutamate receptors. Science 311:1253–1256. doi:10. 1126/science.1123339 Sager C, Tapken D, Kott S, Hollmann M (2009) Functional modulation of AMPA receptors by transmembrane AMPA receptor regulatory proteins. Neuroscience 158:45–54. doi: 10.1016/j.neuroscience.2007.12.046 Sainlos M, Tigaret C, Poujol C et al (2011) Biomimetic divalent ligands for the acute disruption of synaptic AMPAR stabilization. Nat Chem Biol 7:81–91. doi:10. 1038/nchembio.498 Schnell E, Sizemore M, Karimzadegan S et al (2002) Direct interactions between PSD-95 and stargazin control synaptic AMPA receptor number. Proc Natl Acad Sci U S A 99:13902–13907. doi:10.1073/pnas.172511199 Xu W, Schlüter OM, Steiner P et al (2008) Molecular dissociation of the role of PSD-95 in regulating synaptic strength and LTD. Neuron 57:248–262. doi:10.1016/j.neuron.2007.11.027 Hafner A-S, Penn AC, Grillo-Bosch D et al (2015) Lengthening of the stargazin cytoplasmic tail increases synaptic transmission by promoting interaction to deeper domains of PSD-95. Neuron 86(2):475–489. doi:10.1016/j.neuron.2015.03.013 Malinow R, Mainen ZF, Hayashi Y (2000) LTP mechanisms: from silence to four-lane traffic. Curr Opin Neurobiol 10:352–357. doi:10.1016/S0959-4388(00)00099-4 Opazo P, Choquet D (2011) A three-step model for the synaptic recruitment of AMPA receptors. Mol Cell Neurosci 46:1–8. doi: 10.1016/j.mcn.2010.08.014 Sumioka A, Yan D, Tomita S (2010) TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers. Neuron 66:755–767. doi:10.1016/j.neuron.2010.04.035 MacGillavry H, Song Y, Raghavachari S, Blanpied T (2013) Nanoscale scaffolding domains within the postsynaptic density concentrate synaptic AMPA receptors. Neuron 78:615–622. doi: 10.1016/j.neuron.2013.03.009 Garcia RA, Vasudevan K, Buonanno A (2000) The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses. Proc Natl Acad Sci U S A 97:3596–3601. doi:10.1073/pnas.97.7.3596 Huang YZ, Won S, Ali DW et al (2000) Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses. Neuron 26:443–455. doi:10.1016/S0896-6273(00)81176-9 Garcia EP, Mehta S, Blair LAC et al (1998) SAP90 binds and clusters kainate receptors causing incomplete desensitization. Neuron 21:727–739. doi:10.1016/S0896-6273(00)80590-5 Scholl FG, Scheiffele P (2003) Making connections: cholinesterase-domain proteins in the CNS. Trends Neurosci 26: 618–624. doi:10.1016/j.tins.2003.09.004 Niethammer M, Valtschanoff JG, Kapoor TM et al (1998) CRIPT, a novel postsynaptic protein that binds to the third PDZ domain of PSD-95/SAP90. Neuron 20:693–707. doi: 10.1016/S0896-6273(00)81009-0 Saro D, Li T, Rupasinghe C et al (2007) A thermodynamic ligand binding study of the third PDZ domain (PDZ3) from the mammalian neuronal protein PSD-95. Biochemistry 46:6340–6352. doi: 10.1021/bi062088k Fukata Y, Adesnik H, Iwanaga T et al (2006) Epilepsy-related ligand/receptor complex LGI1 and ADAM22 regulate synaptic transmission. Science 313:1792–1795. doi:10.1126/science. 1129947 Kim E, Niethammer M, Rothschild A et al (1995) Clustering of Shaker-type K+ channels by interaction with a family of

64.

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

membrane-associated guanylate kinases. Nature 378:85–88. doi: 10.1038/378085a0 Cohen NA, Brenman JE, Snyder SH, Bredt DS (1996) Binding of the inward rectifier K+ channel Kir 2.3 to PSD-95 is regulated by protein kinase A phosphorylation. Neuron 17:759–767. doi:10. 1016/S0896-6273(00)80207-X Firestein BL, Firestein BL, Brenman JE et al (1999) Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting. Neuron 24: 659–672 Akum BF, Chen M, Gunderson SI et al (2004) Cypin regulates dendrite patterning in hippocampal neurons by promoting microtubule assembly. Nat Neurosci 7:145–152. doi:10.1038/nn1179 Hillier BJ, Christopherson KS, Prehoda KE et al (1999) Unexpected modes of PDZ domain scaffolding revealed by structure of nNOS-syntrophin complex. Science 284:812–815. doi:10. 1126/science.284.5415.812 Gardoni F, Polli F, Cattabeni F, Di Luca M (2006) Calciumcalmodulin-dependent protein kinase II phosphorylation modulates PSD-95 binding to NMDA receptors. Eur J Neurosci 24: 2694–2704. doi:10.1111/j.1460-9568.2006.05140.x Tezuka T, Umemori H, Akiyama T et al (1999) PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the N-methyl-Daspartate receptor subunit NR2A. Proc Natl Acad Sci U S A 96: 435–440. doi:10.1073/pnas.96.2.435 Songyang Z, Fanning AS, Fu C et al (1997) Recognition of unique carboxyl-terminal motifs by distinct PDZ domains. Science 275: 73–77. doi:10.1126/science.275.5296.73 Takeuchi M, Hata Y, Hirao K et al (1997) SAPAPs. A family of PSD-95/SAP90-associated proteins localized at postsynaptic density. J Biol Chem 272:11943–11951. doi:10.1074/jbc.272.18. 11943 Tao-Cheng J-H, Yang Y, Reese TS, Dosemeci A (2015) Differential distribution of Shank and GKAP at the postsynaptic density. PLoS One 10:e0118750. doi:10.1371/journal.pone. 0118750 Reese ML, Dakoji S, Bredt DS, Dötsch V (2007) The guanylate kinase domain of the MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a. Nat Struct Mol Biol 14:155–163. doi: 10.1038/nsmb1195 Szebenyi G, Bollati F, Bisbal M et al (2005) Activity-driven dendritic remodeling requires microtubule-associated protein 1A. Curr Biol 15:1820–1826. doi:10.1016/j.cub.2005.08.069 Kim JH, Liao D, Lau LF, Huganir RL (1998) SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family. Neuron 20:683–691. doi:10.1016/S0896-6273(00)81008-9 Naisbitt S, Valtschanoff J, Allison DW et al (2000) Interaction of the postsynaptic density-95/guanylate kinase domain-associated protein complex with a light chain of myosin-V and dynein. J Neurosci 20:4524–4534 Charych EI, Akum BF, Goldberg JS et al (2006) Activityindependent regulation of dendrite patterning by postsynaptic density protein PSD-95. J Neurosci 26:10164–10176. doi:10. 1523/JNEUROSCI.2379-06.2006 Zhao J-P, Murata Y, Constantine-Paton M (2013) Eye opening and PSD95 are required for long-term potentiation in developing superior colliculus. Proc Natl Acad Sci U S A 110:707–712. doi:10. 1073/pnas.1215854110 Ehrlich I, Malinow R (2004) Postsynaptic density 95 controls AMPA receptor incorporation during long-term potentiation and experience-driven synaptic plasticity. J Neurosci 24:916–927. doi: 10.1523/JNEUROSCI.4733-03.2004 El-Husseini AE, Schnell E, Chetkovich DM et al (2000) PSD-95 involvement in maturation of excitatory synapses. Science 290: 1364–1368. doi:10.1126/science.290.5495.1364 van Zundert B, Yoshii A, Constantine-Paton M (2004) Receptor compartmentalization and trafficking at glutamate synapses: a

Mol Neurobiol

82.

83.

84.

85.

86.

87.

88.

89.

90.

91.

92. 93.

94.

95.

96.

97. 98.

99.

100.

developmental proposal. Trends Neurosci 27:428–437. doi:10. 1016/j.tins.2004.05.010 Bustos FJ, Varela-Nallar L, Campos M et al (2014) PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors. PLoS One 9:e94037. doi:10.1371/journal.pone.0094037 Henriquez B, Bustos FJ, Aguilar R et al (2013) Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons. Mol Cell Neurosci 57:130–143. doi:10. 1016/j.mcn.2013.07.012 Ehrlich I, Klein M, Rumpel S, Malinow R (2007) PSD-95 is required for activity-driven synapse stabilization. Proc Natl Acad Sci U S A 104:4176–4181. doi:10.1073/pnas.0609307104 Yoshii A, Sheng MH, Constantine-Paton M (2003) Eye opening induces a rapid dendritic localization of PSD-95 in central visual neurons. Proc Natl Acad Sci U S A 100:1334–1339. doi:10.1073/ pnas.0335785100 Yoshii A, Constantine-Paton M (2007) BDNF induces transport of PSD-95 to dendrites through PI3K-AKT signaling after NMDA receptor activation. Nat Neurosci 10:702–711. doi:10.1038/ nn1903 Mumby SM (1997) Reversible palmitoylation of signaling proteins. Curr Opin Cell Biol 9:148–154. doi:10.1016/S09550674(97)80056-7 Sunyer B, Diao W, Lubec G (2008) The role of post-translational modifications for learning and memory formation. Electrophoresis 29:2593–2602. doi:10.1002/elps.200700791 Chamberlain LH, Shipston MJ (2015) The physiology of protein S-acylation. Physiol Rev 95:341–376. doi:10.1152/physrev. 00032.2014 Topinka JR, Bredt DS (1998) N-terminal palmitoylation of PSD95 regulates association with cell membranes and interaction with K+ channel K(v)1.4. Neuron 20:125–134. doi:10.1016/S08966273(00)80440-7 Craven SE, El-Husseini AE, Bredt DS (1999) Synaptic targeting of the postsynaptic density protein PSD-95 mediated by lipid and protein motifs. Neuron 22:497–509. doi:10.1074/jbc. M910153199 O’Brien PJ, Zatz M (1984) Acylation of bovine rhodopsin by [3H]palmitic acid. J Biol Chem 259:5054–5057 Sudo Y, Valenzuela D, Beck-Sickinger AG et al (1992) Palmitoylation alters protein activity: blockade of G(o) stimulation by GAP-43. EMBO J 11:2095–2102 Wedegaertner PB, Chu DH, Wilson PT et al (1993) Palmitoylation is required for signaling functions and membrane attachment of Gqα and Gsα. J Biol Chem 268:25001–25008 Niethammer P, Delling M, Sytnyk V et al (2002) Cosignaling of NCAM via lipid rafts and the FGF receptor is required for neuritogenesis. J Cell Biol 157:521–532. doi:10.1083/jcb. 200109059 Fukata Y, Fukata M (2010) Protein palmitoylation in neuronal development and synaptic plasticity. Nat Rev Neurosci 11:161– 175. doi:10.1038/nrn2788 Fukata M, Fukata Y, Adesnik H et al (2004) Identification of PSD95 palmitoylating enzymes. Neuron 44:987–996 Fukata Y, Iwanaga T, Fukata M (2006) Systematic screening for palmitoyl transferase activity of the DHHC protein family in mammalian cells. Methods 40:177–182. doi:10.1016/j.ymeth. 2006.05.015 Noritake J, Fukata Y, Iwanaga T et al (2009) Mobile DHHC palmitoylating enzyme mediates activity-sensitive synaptic targeting of PSD-95. J Cell Biol 186:147–160. doi:10.1083/jcb. 200903101 El-Husseini AED, Schnell E, Dakoji S et al (2002) Synaptic strength regulated by palmitate cycling on PSD-95. Cell 108: 849–863. doi:10.1016/S0092-8674(02)00683-9

101.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

115.

116. 117. 118.

119.

Fukata Y, Dimitrov A, Boncompain G et al (2013) Local palmitoylation cycles define activity-regulated postsynaptic subdomains. J Cell Biol 202:145–161. doi:10.1083/jcb. 201302071 El-Husseini AE, Bredt DS (2002) Protein palmitoylation: a regulator of neuronal development and function. Nat Rev Neurosci 3: 791–802. doi:10.1038/nrn940 Li B, Otsu Y, Murphy TH, Raymond LA (2003) Developmental decrease in NMDA receptor desensitization associated with shift to synapse and interaction with postsynaptic density-95. J Neurosci 23:11244–11254 Yoshii A, Murata Y, Kim J et al (2011) TrkB and protein kinase M {zeta} regulate synaptic localization of PSD-95 in developing cortex. J Neurosci 31:11894–11904. doi:10.1523/JNEUROSCI. 2190-11.2011 Parsons MP, Kang R, Buren C et al (2014) Bidirectional control of postsynaptic density-95 (PSD-95) clustering by Huntingtin. J Biol Chem 289:3518–3528. doi:10.1074/jbc.M113.513945 Yoshii A, Constantine-Paton M (2014) Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling. Front Synaptic Neurosci 6:1–7. doi:10.3389/fnsyn. 2014.00006 Zhang Y, Matt L, Patriarchi T et al (2014) Capping of the Nterminus of PSD-95 by calmodulin triggers its postsynaptic release. EMBO J 33:1341–1353. doi:10.1002/embj.201488126 Stamler JS, Simon DI, Osborne JA et al (1992) S-nitrosylation of proteins with nitric oxide: synthesis and characterization of biologically active compounds. Proc Natl Acad Sci U S A 89:444– 448 Nakamura T, Lipton SA (2015) Nitrosative stress in the nervous system: guidelines for designing experimental strategies to study protein S-nitrosylation. Neurochem Res. doi:10.1007/s11064015-1640-z Nakamura T, Tu S, Akhtar M et al (2013) Aberrant protein Snitrosylation in neurodegenerative diseases. Neuron 78:596–614. doi:10.1016/j.neuron.2013.05.005 Hess DT, Matsumoto A, Kim S-O et al (2005) Protein Snitrosylation: purview and parameters. Nat Rev Mol Cell Biol 6: 150–166. doi:10.1038/nrm1569 Whalen EJ, Foster MW, Matsumoto A et al (2007) Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-proteincoupled receptor kinase 2. Cell 129:511–522. doi:10.1016/j.cell. 2007.02.046 Ho GPH, Selvakumar B, Mukai J et al (2011) S-nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD95. Neuron 71:131–141. doi:10.1016/j.neuron.2011.05.033 Van Weeren PC, De Bruyn KMT, De Vries-Smits AMM et al (1998) Essential role for protein kinase B (PKB) in insulininduced glycogen synthase kinase 3 inactivation. Characterization of dominant-negative mutant of PKB. J Biol Chem 273:13150–13156. doi:10.1074/jbc.273.21.13150 Cole PA, Shen K, Qiao Y, Wang D (2003) Protein tyrosine kinases Src and Csk: a tail’s tale. Curr Opin Chem Biol 7:580–585. doi:10. 1016/j.cbpa.2003.08.009 Babior BM (1999) NADPH oxidase: an update. Blood 93:1464– 1476 Cohen P (2002) The origins of protein phosphorylation. Nat Cell Biol 4:E127–E130. doi:10.1038/ncb0502-e127 Weeks ACW, Ivanco TL, Leboutillier JC et al (2000) Sequential changes in the synaptic structural profile following long- term potentiation in the rat dentate gyrus. II. Induction/early maintenance phase. Synapse 36:286–296. doi:10.1002/(SICI)10982396(20000615)36:43.0.CO;2-T Dosemeci A, Tao-Cheng JH, Vinade L et al (2001) Glutamateinduced transient modification of the postsynaptic density. Proc

Mol Neurobiol

120.

121.

122.

123.

124.

125.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

Natl Acad Sci U S A 98:10428–10432. doi:10.1073/pnas. 181336998 Ehlers MD (2003) Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasome system. Nat Neurosci 6:231–242. doi:10.1038/nn1013 Uematsu K, Heiman M, Zelenina M et al (2015) Protein kinase A directly phosphorylates metabotropic glutamate receptor 5 to modulate its function. J Neurochem 132:677–686. doi:10.1111/ jnc.13038 Chergui K, Svenningsson P, Greengard P (2005) Physiological role for casein kinase 1 in glutamatergic synaptic transmission. J Neurosci 25:6601–6609. doi:10.1523/JNEUROSCI.1082-05. 2005 Greengard P (2001) The neurobiology of slow synaptic transmission. Science 294:1024–1030. doi:10.1126/science.294.5544. 1024 de Arce KP, Varela-Nallar L, Farias O et al (2010) Synaptic clustering of PSD-95 is regulated by c-Abl through tyrosine phosphorylation. J Neurosci 30:3728–3738. doi:10.1523/JNEUROSCI. 2024-09.2010 Du C-P, Gao J, Tai J-M et al (2009) Increased tyrosine phosphorylation of PSD-95 by Src family kinases after brain ischaemia. Biochem J 417:277–285. doi:10.1042/BJ20080004 Zhao C, Du C-P, Peng Y et al (2015) The upregulation of NR2Acontaining N-methyl-D-aspartate receptor function by tyrosine phosphorylation of postsynaptic density 95 via facilitating Src/ proline-rich tyrosine kinase 2 activation. Mol Neurobiol 51:500– 511. doi:10.1007/s12035-014-8796-4 Morabito MA, Sheng M, Tsai L-H (2004) Cyclin-dependent kinase 5 phosphorylates the N-terminal domain of the postsynaptic density protein PSD-95 in neurons. J Neurosci 24:865–876. doi: 10.1523/JNEUROSCI.4582-03.2004 Chung HJ, Huang YH, Lau L-F, Huganir RL (2004) Regulation of the NMDA receptor complex and trafficking by activitydependent phosphorylation of the NR2B subunit PDZ ligand. J Neurosci 24:10248–10259. doi:10.1523/JNEUROSCI.0546-04. 2004 Soto D, Pancetti F, Marengo JJ et al (2004) Protein kinase CK2 in postsynaptic densities: phosphorylation of PSD-95/SAP90 and NMDA receptor regulation. Biochem Biophys Res Commun 322:542–550. doi:10.1016/j.bbrc.2004.07.158 Jaffe H, Vinade L, Dosemeci A (2004) Identification of novel phosphorylation sites on postsynaptic density proteins. Biochem Biophys Res Commun 321:210–218. doi:10.1016/j.bbrc.2004.06. 122 Kim MJ, Futai K, Jo J et al (2007) Synaptic accumulation of PSD95 and synaptic function regulated by phosphorylation of serine295 of PSD-95. Neuron 56:488–502. doi:10.1016/j.neuron.2007. 09.007 Sabio G, Reuver S, Feijoo C et al (2004) Stress- and mitogeninduced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2. Biochem J 380:19–30. doi:10.1042/BJ20031628 Nelson CD, Kim MJ, Hsin H et al (2013) Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression. J Neurosci 33:12122–12135. doi:10.1523/JNEUROSCI.0131-13.2013 Tsui J, Malenka RC (2006) Substrate localization creates specificity in calcium/calmodulin-dependent protein kinase II signaling at synapses. J Biol Chem 281:13794–13804. doi:10.1074/jbc. M600966200 Steiner P, Higley MJ, Xu W et al (2008) Destabilization of the postsynaptic density by PSD-95 serine 73 phosphorylation inhibits spine growth and synaptic plasticity. Neuron 60:788–802. doi:10.1016/j.neuron.2008.10.014

136.

137.

138. 139. 140.

141.

142.

143.

144.

145.

146. 147.

148.

149.

150.

151.

152.

153.

154.

155.

156.

Glaunsinger BA, Lee SS, Thomas M et al (2000) Interactions of the PDZ-protein MAGI-1 with adenovirus E4-ORF1 and highrisk papillomavirus E6 oncoproteins. Oncogene 19:5270–5280. doi:10.1038/sj.onc.1203906 DiAntonio A, Haghighi AP, Portman SL et al (2001) Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412:449–452. doi:10.1038/35086595 Hegde AN, DiAntonio A (2002) Ubiquitin and the synapse. Nat Rev Neurosci 3:854–861. doi:10.1038/nrn961 Yi JJ, Ehlers MD (2005) Ubiquitin and protein turnover in synapse function. Neuron 47:629–632. doi:10.1016/j.neuron.2005.07.008 Yi JJ, Ehlers MD (2007) Emerging roles for ubiquitin and protein degradation in neuronal function. Pharmacol Rev 59:14–39. doi: 10.1124/pr.59.1.4 Hershko A, Heller H, Elias S, Ciechanover A (1983) Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem 258:8206–8214 Deveraux Q, Ustrell V, Pickart C, Rechsteiner M (1994) A 26 S protease subunit that binds ubiquitin conjugates. J Biol Chem 269: 7059–7061 Ciechanover A, Elias S, Heller H, Hershko A (1982) “Covalent affinity” purification of ubiquitin-activating enzyme. J Biol Chem 257:2537–2542 Hershko A, Ciechanover A, Rose IA (1981) Identification of the active amino acid residue of the polypeptide of ATP-dependent protein breakdown. J Biol Chem 256:1525–1528 Pierce NW, Kleiger G, Shan S, Deshaies RJ (2009) Detection of sequential polyubiquitylation on a millisecond timescale. Nature 462:615–619. doi:10.1038/nature08595 Weissman AM (2001) Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2:169–178. doi:10.1038/35056563 Waataja JJ, Kim HJ, Roloff AM, Thayer SA (2008) Excitotoxic loss of post-synaptic sites is distinct temporally and mechanistically from neuronal death. J Neurochem 104:364–375. doi:10. 1111/j.1471-4159.2007.04973.x Colledge M, Snyder EM, Crozier RA et al (2003) Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression. Neuron 40:595–607. doi:10.1016/S0896-6273(03)00687-1 Bianchetta MJ, Lam TT, Jones SN, Morabito MA (2011) Cyclindependent kinase 5 regulates PSD-95 ubiquitination in neurons. J Neurosci 31:12029–12035. doi:10.1523/JNEUROSCI.2388-11. 2011 Opazo CM, Greenough MA, Bush AI (2014) Copper: from neurotransmission to neuroproteostasis. Front Aging Neurosci. doi: 10.3389/fnagi.2014.00143 Park D, Ravichandran KS (2010) Emerging roles of brain-specific angiogenesis inhibitor 1. Adv Exp Med Biol 706:167–178. doi:10. 1007/978-1-4419-7913-1_15 Cork SM, Van Meir EG (2011) Emerging roles for the BAI1 protein family in the regulation of phagocytosis, synaptogenesis, neurovasculature, and tumor development. J Mol Med 89:743– 752. doi:10.1007/s00109-011-0759-x Mori K, Kanemura Y, Fujikawa H et al (2002) Brain-specific angiogenesis inhibitor 1 (BAI1) is expressed in human cerebral neuronal cells. Neurosci Res 43:69–74. doi:10.1016/S01680102(02)00018-4 Zhu D, Li C, Swanson AM et al (2015) BAI1 regulates spatial learning and synaptic plasticity in the hippocampus. J Clin Invest 125:1–12. doi:10.1172/JCI74603) Herrmann J, Lerman LO, Lerman A (2007) Ubiquitin and ubiquitin-like proteins in protein regulation. Circ Res 100:1276– 1291. doi:10.1161/01.RES.0000264500.11888.f0 Rabut G, Peter M (2008) Function and regulation of protein neddylation. “Protein modifications: beyond the usual suspects” review series. EMBO Rep 9:969–976. doi:10. 1038/embor.2008.183

Mol Neurobiol 157.

Vogl AM, Brockmann MM, Giusti SA et al (2015) Neddylation inhibition impairs spine development, destabilizes synapses and deteriorates cognition. Nat Neurosci 18: 239–251. doi:10.1038/nn.3912 158. Fu AK, Ip NY (2015) Neddylation is needed for synapse maturation. Nat Publ Group 18:164–166. doi:10.1038/nn. 3929 159. Dinamarca MC, Ríos JA, Inestrosa NC (2012) Postsynaptic receptors for amyloid-β oligomers as mediators of neuronal damage in Alzheimer’s disease. Front Physiol. doi:10.3389/fphys.2012. 00464 160. Dinamarca MC, Di Luca M, Godoy JA, Inestrosa NC (2015) The soluble extracellular fragment of neuroligin-1 targets Aβ oligomers to the postsynaptic region of excitatory synapses. Biochem Biophys Res Commun 466:66–71. doi:10.1016/j.bbrc.2015.08. 107 161. Dinamarca MC, Weinstein D, Monasterio O, Inestrosa NC (2011) The synaptic protein neuroligin-1 interacts with the amyloid βpeptide. Is there a role in Alzheimer’s disease? Biochemistry 50: 8127–8137. doi:10.1021/bi201246t 162. Lacor PN, Buniel MC, Chang L et al (2004) Synaptic targeting by Alzheimer’s-related amyloid beta oligomers. J Neurosci 24: 10191–10200. doi:10.1523/JNEUROSCI.3432-04.2004 163. Pham E, Crews L, Ubhi K et al (2010) Progressive accumulation of amyloid-beta oligomers in Alzheimer’s disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins. FEBS J 277:3051–3067. doi:10.1111/j.1742-4658.2010.07719.x 164. Savioz A, Leuba G, Vallet PG (2014) A framework to understand the variations of PSD-95 expression in brain aging and in Alzheimer’s disease. Ageing Res Rev 18:86–94. doi:10.1016/j. arr.2014.09.004 165. Roselli F, Tirard M, Lu J et al (2005) Soluble beta-amyloid1-40 induces NMDA-dependent degradation of postsynaptic density95 at glutamatergic synapses. J Neurosci 25:11061–11070. doi: 10.1523/JNEUROSCI.3034-05.2005 166. Snyder EM, Nong Y, Almeida CG et al (2005) Regulation of NMDA receptor trafficking by amyloid-beta. Nat Neurosci 8: 1051–1058. doi:10.1038/nn1503 167. Hsieh H, Boehm J, Sato C et al (2006) AMPAR removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron 52:831–843. doi:10.1016/j.neuron.2006. 10.035 168. Codocedo JF, Montecinos-Oliva C, Inestrosa NC (2015) Wntrelated SynGAP1 is a neuroprotective factor of glutamatergic synapses against Aβ oligomers. Front Cell Neurosci 9:227. doi:10. 3389/fncel.2015.00227 169. Cerpa W, Farías GG, Godoy JA et al (2010) Wnt-5a occludes Abeta oligomer-induced depression of glutamatergic transmission in hippocampal neurons. Mol Neurodegener 5: 3. doi:10.1186/1750-1326-5-3 170. Ittner LM, Ke YD, Delerue F et al (2010) Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer’s disease mouse models. Cell 142:387–397. doi:10.1016/j. cell.2010.06.036 171. Young FB, Butland SL, Sanders SS et al (2012) Putting proteins in their place: palmitoylation in Huntington disease and other neuropsychiatric diseases. Prog Neurobiol 97:220–238. doi:10.1016/j. pneurobio.2011.11.002 172. Raymond FL, Tarpey PS, Edkins S et al (2007) Mutations in ZDHHC9, which encodes a palmitoyltransferase of NRAS and HRAS, cause X-linked mental retardation associated with a Marfanoid habitus. Am J Hum Genet 80:982– 987. doi:10.1086/513609 173. Mansouri MR, Marklund L, Gustavsson P et al (2005) Loss of ZDHHC15 expression in a woman with a balanced translocation

174.

175.

176.

177.

178.

179.

180.

181.

182. 183.

184.

185.

186.

187.

188.

189.

190.

t(X;15)(q13.3;cen) and severe mental retardation. Eur J Hum Genet 13:970–977. doi:10.1038/sj.ejhg.5201445 Mizumaru C, Saito Y, Ishikawa T et al (2009) Suppression of APP-containing vesicle trafficking and production of β-amyloid by AID/DHHC-12 protein. J Neurochem 111:1213–1224. doi:10. 1111/j.1471-4159.2009.06399.x Chen WY, Shi YY, Zheng YL et al (2004) Case-control study and transmission disequilibrium test provide consistent evidence for association between schizophrenia and genetic variation in the 22q11 gene ZDHHC8. Hum Mol Genet 13:2991–2995. doi:10. 1093/hmg/ddh322 Mukai J, Liu H, Burt RA et al (2004) Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia. Nat Genet 36:725–731. doi:10.1038/ng1375 Mukai J, Dhilla A, Drew LJ et al (2008) Palmitoylation-dependent neurodevelopmental deficits in a mouse model of 22q11 microdeletion. Nat Neurosci 11:1302–1310. doi:10.1038/nn.2204 Huang K, Yanai A, Kang R et al (2004) Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins. Neuron 44:977–986. doi:10.1016/j.neuron.2004.11.027 Yanai A, Huang K, Kang R et al (2006) Palmitoylation of Huntingtin by HIP14 is essential for its trafficking and function. Nat Neurosci 9:824–831. doi:10.1038/nn1702 Flavell SW, Cowan CW, Kim T-K et al (2006) Activity-dependent regulation of MEF2 transcription factors suppresses excitatory synapse number. Science 311:1008–1012. doi:10.1126/science. 1122511 Pfeiffer BE, Zang T, Wilkerson JR et al (2010) Fragile X mental retardation protein is required for synapse elimination by the activity-dependent transcription factor MEF2. Neuron 66:191– 197. doi:10.1016/j.neuron.2010.03.017 Kelleher RJ, Bear MF (2008) The autistic neuron: troubled translation? Cell 135:401–406. doi:10.1016/j.cell.2008.10.017 Tsai NP, Wilkerson JR, Guo W et al (2012) Multiple autism-linked genes mediate synapse elimination via proteasomal degradation of a synaptic scaffold PSD-95. Cell 151:1581–1594. doi:10.1016/j. cell.2012.11.040 Frederikse PH, Nandanoor A, Kasinathan C (2015) Fragile X syndrome FMRP co-localizes with regulatory targets PSD-95, GABA receptors, CaMKIIα, and mGluR5 at fiber cell membranes in the eye lens. Neurochem Res. doi:10.1007/s11064-015-1702-2 Nissen KB, Andersen JJ, Haugaard-Kedström LM et al (2015) Design, synthesis, and characterization of fatty acid derivatives of a dimeric peptide-based postsynaptic density-95 (PSD-95) inhibitor. J Med Chem 58:1575–1580. doi:10.1021/jm501755d Nissen KB, Haugaard-Kedström LM, Wilbek TS et al (2015) Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family. PLoS One 10: e0117668. doi:10.1371/journal.pone.0117668 Bach A, Clausen BH, Moller M et al (2012) A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage. Proc Natl Acad Sci 109: 3317–3322. doi:10.1073/pnas.1113761109 LeBlanc BW, Iwata M, Mallon AP et al (2010) A cyclic peptide targeted against PSD-95 blocks central sensitization and attenuates thermal hyperalgesia. Neuroscience 167:490–500. doi:10.1016/j. neuroscience.2010.02.031 Tao F, Su Q, Johns RA (2008) Cell-permeable peptide Tat-PSD-95 PDZ2 inhibits chronic inflammatory pain behaviors in mice. Mol Ther 16:1776–1782. doi:10.1038/mt.2008.192 Ha CM, Park D, Han JK et al (2012) Calcyon forms a novel ternary complex with dopamine D1 receptor through PSD-95 protein and plays a role in dopamine receptor internalization. J Biol Chem 287:31813–31822. doi:10.1074/jbc.M112.370601