Postsynaptic density scaffolding proteins at excitatory synapse and disorders of synaptic plasticity: implications for human behavior pathologies

POSTSYNAPTIC DENSITY SCAFFOLDING PROTEINS AT EXCITATORY SYNAPSE AND DISORDERS OF SYNAPTIC PLASTICITY: IMPLICATIONS FOR HUMAN BEHAVIOR PATHOLOGIES

Andrea de Bartolomeis and Germano Fiore Laboratory of Molecular Psychiatry Department of Neuroscience and Behavioral Sciences University Medical School of Naples, Federico II, Italy

I. Introduction II. Structural and Functional Organization of Postsynaptic Density (PSD) Proteins: An Overview A. ScaVolding Proteins B. MAGUK Proteins C. Protein Kinases and Protein Phosphatases D. Receptors E. PSD Proteins and the Concept of Microdomain F. PSD Proteins and Receptor DiVusion III. PSD-95/SAP90 A. Structure B. Functions C. PSD-95/SAP90 and Neuropsychiatric Disorders IV. Shank/ProSAP Proteins A. Structure B. Functions C. Shank3 and the 22q13.3 Deletion Syndrome V. SAP97 A. Structure B. Functions C. SAP97 and Schizophrenia VI. Homer Proteins A. Structure B. Functions C. Homer 1a Induction and Implication for Neuropsychiatric Disorders VII. Conclusive Remarks References

Excitatory synapses are characterized by an electron-dense thickening at the cytoplasmic surface of the postsynaptic membrane, called the postsynaptic density (PSD). The PSD is a fibrous specialization of the submembrane cytoskeleton approximately 30–40 nm thick and about 100 nm wide. Hundreds of molecules have been identified in the PSD: ion-gated and G-protein-coupled receptors, association, adaptors, and scaVolding proteins, key enzymes involved INTERNATIONAL REVIEW OF NEUROBIOLOGY, VOL. 59

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in phosphorylation–dephosphorylation mechanisms, and cytoskeletal proteins. Each of these proteins may have a pivotal function in setting the molecular scenario for the development of synaptic plasticity. ScaVolding proteins are major players in the organization of the postsynaptic signal transduction machinery, they regulate receptor traYcking and clustering, modulate axon pathfinding, and drive the correct targeting of neuronal proteins to their appropriate cytoplasmic compartment. Emerging findings suggest a relevant involvement of PSD scaVolding/adaptor proteins in behavior modulation in animal models of synaptic plasticity disorders and pharmacological isomorphisms.

I. Introduction

Synaptic plasticity can be viewed as the result of a highly regulated form of neuronal communication (Bear and Malenka, 1994; Sheng and Kim, 2002). Among the structures associated with the chemical synapse, postsynaptic density (PSD) has recently attracted special interest for the sophisticated mechanisms of molecule interaction and regulation of signal transduction that it shows (Kennedy, 1997, 2000; Scannevin and Huganir, 2000). Postsynaptic density at the excitatory synapse is a disc-shaped structure that can be recognized under electron microscopy as an organelle of about 50 nm thick, localized beneath the postsynaptic membrane of the type I synapse (axodentric synapse) prominently and facing the presynaptic active zone (Kennedy, 2000). The PSD can be purified from brain tissue by diVerential centrifugation, and several proteins have been identified by means of mass spectrometry (Yamauchi, 2002). Ionic and G-protein-coupled glutamate receptors, scaVolding proteins, adaptor and receptor interactor proteins, kinases and phosphatases, key enzymes, and cytoskeleton proteins have been described at the PSD (Garner et al., 2000; Kennedy, 1998; Sheng and Pak, 2000; Yamauchi, 2002; ZiV, 1997), constituting a dynamic protein lattice (Scannevin and Huganir, 2000) with a highly regulated molecular organization. PSD proteins are thought to play a major role in neurodevelopment (Foa et al., 2001), dendritic spine formation (Sala et al., 2001), receptor electrophysiological property regulation (Yamada et al., 1999a,b), receptor clustering (Ehlers et al., 1996; Kornau et al., 1997; Sheng, 1996; Sheng and Kim, 2002), and linking membrane receptors directly to second messenger signaling (Xiao et al., 1998). Moreover, findings have shown a putative role for PSD proteins in human behavior pathologies and potential implications in pharmacotherapy (de Bartolomeis et al., 2002; Nesslinger et al., 1994; Ohnuma et al., 2000; Prasad et al., 2000). All the major components of the PSD have been implicated directly or indirectly in synaptic plasticity regulation; it is beyond the scope of this review to describe the massive amount of findings on the role of PSD

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proteins in neuronal function (Sheng and Kim, 2002). This review focuses on PSD scaVolding proteins at the glutamatergic postsynaptic density that have been demonstrated to be involved specifically in disorders of synaptic plasticity, with special emphasis on those proteins for which data are available linking them with human behavior pathologies and, possibly, with their treatment. Nevertheless, animal models and pharmacologic isomorphisms are considered when pertinent to better understand the pathophysiology of synaptic plasticity disorders in humans.

II. Structural and Functional Organization of Postsynaptic Density (PSD) Proteins: An Overview

The PSD at the excitatory synapse is a hierarchically and highly dynamic organized structure where signal transduction is processed, modified, integrated, and propagated. Proteins of the PSD may be classified according to their function in scaVolding, association, adhesion, cytoskeletal elements, and as kinase and phosphatase enzymes (Yamauchi, 2002). Each PSD protein may have a pivotal function in setting the scenario for the development of synaptic plasticity, and emerging findings point to an implication of PSD proteins in behavior modulation in animal models, as well as in pharmacological isomorphisms.

A. Scaffolding Proteins Among PSD molecules believed to have a direct role in synaptic plasticity, scaVolding proteins deserve special attention for their multifunctional property of adaptor proteins (ZiV, 1997). ScaVolding proteins may influence the function and availability of receptors by clustering and connecting them physically with other pivotal PSD proteins as well as with intracytoplasmic molecules. Considering the high concentration of glutamate receptors of ionic type [amino-3-hydroxy-5methyl-4-isoxazole propionate (AMPA) and N-methyl-d-aspartate (NMDA) receptors] and metabotropic type (mGluR) at PSD of excitatory synapses, the correct alignment and the precise spatial distribution of these receptors, as well as the specific interaction with intracytoplasmic proteins responsible for integrating the signal transduction, are crucial requirements. Postsynaptic density 95/synapse-associated protein 90 (PSD-95/SAP90) (Cho et al., 1992; Kistner et al., 1993), Homer family (Brakeman et al., 1997; Xiao et al., 1998), and glutamate receptor interacting protein (GRIP) (Dong et al., 1997, 1999) are the major postsynaptic density proteins at glutamatergic synapse with a

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scaVolding function and interacting with the NMDA NR2 subunit, type I mGluR (mGluR1 and mGluR5) and AMPA receptors (AMPA-R), respectively. It is worth emphasizing that, based on the nomenclature of glutamate receptorassociated proteins, the scaVolding proteins may be also classified as glutamate receptor interactors, a group including several other molecules regulating receptor signal transduction but with no fully demonstrated scaVolding function, such as Yotiao (Lin et al., 1998), AMPA receptor-binding protein (ABP) (Srivastava et al., 1998), mammalian Lin seven (MALS) ( Jo et al., 1999), neural activity-regulated pentatraxin (NARP) (O’Brien et al., 1999), and protein interacting with C kinase (PICK 1) (Xia et al., 1999). Finally, protein members of the same family of PSD proteins may have diVerent functions; however, a clear dissection between scaVolding and anchoring or adaptor function may not be clear-cut. This is the case in the Homer family with Homer 1b included in the scaVolding group of proteins and Homer 1c demonstrated to have a scaVolding but also anchoring function (Ciruela et al., 2000).

B. MAGUK Proteins Based on gene structure and domain interaction specificity, some scaVolding proteins also belong to a large group of membrane-associated proteins called membrane-associated guanylate kinase (MAGUK) proteins (Niethammer et al., 1996). This group includes, among others, PSD 95/SAP90 (Cho et al., 1992), PSD 93/chapsyn-110 (Kim et al., 1996), SAP 97 (Muller et al., 1995), and SAP 102. In addition to the guanylate kinase domain, MAGUK proteins contain a SH3 domain and three PDZ domains (see Section III). The complexity of MAGUK protein structure may explain the multiple modulator functions of these groups of proteins demonstrated to be involved not only in receptor clustering, but also in regulation of the electrophysiological properties of the receptor (Yamada et al., 1999a), as well as in linking glutamate receptors directly to second messenger signaling systems (Brenman et al., 1996a).

C. Protein Kinases and Protein Phosphatases Several kinase proteins have been identified at the PSD: calcium/calmodulindependent protein kinase type II (CaMKII), protein kinases A and C, MAP kinase, the Src family tyrosine kinase, the trkB receptor tyrosine kinase, and the Erb receptor tyrosine kinase. It is important to note that the same PSD proteins may be a substrate of diVerent kinases. For example, dynamin may be phosphorylated by PKA, Src family tyrosine kinase, or MAP kinase.

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CaMKII is the most abundant kinase at PSD. Four diVerent isoforms (, ,

, and  ) are known to aggregate to form a dodecameric enzyme. The functionally active form of the kinase in neurons is represented by the  and subunits, with the  subunit largely prevailing in the holoenzyme over the subunit. CaMKII may undergo autophosphorylation, followed by translocation to the PSD where the kinase is believed to enhance the synaptic strength by phosphorylating ion channels and receptors. ScaVolding, association, and interactor, as well as cytoskeleton PSD proteins, are substrates for the CaMKII. PSD 95/SAP90, SAP 97, guanylate kinase-associated protein (GKAP), rasGTPase-activating protein, and synaptic GTPase-activating protein (SynGAP) family proteins have several consensus sequences of the phosphorylation site RXXXS/T. Even the precise function of CaMKII-dependent phosphorylation of diVerent PSD proteins has yet to be unraveled. It is thought to represent a relevant mechanism in the maintenance of dendritic architecture and synaptic plasticity (Means, 2000). The following sequence of events has been suggested: the release of neurotransmitter at presynaptic terminal triggers the Ca2þ influx through the NMDA and AMPA receptors, and calcium activates the postsynaptic CaMKII, which translocates from the cytosol to the postsynaptic density and phosphorylates (activating or inhibiting) several PSD proteins (Soderling, 2000). For example, the phosphorylation and inhibition of SynGAP may result in potentiation of the mitogen-activated protein kinase pathway (Chen et al., 1998; Kim et al., 1998).

D. Receptors NMDA, kainate, AMPA, and metabotropic aglutamate receptors have all been identified in the PSD glutamatergic synapse. With regard to the receptor– scaVolding proteins interaction, the NMDA receptor (NMDA-R) may be considered to be the core of the PSD based on its role, together with AMPA-Rs, in long-term potentiation (LTP), considered a highly regulated process of synaptic plasticity. LTP has been reported to be impaired by the disruption of normal PSD scaVolding protein function (Migaud et al., 1998). Several proteins at the PSD have been demonstrated to interact with NMDA-Rs; moreover, this receptor binds directly at least two PSD scaVolding and anchoring proteins, PSD-95/ SAP90 and Yotiao, through, respectively, the NR2 and NR1 subunits. Metabotropic glutamate receptors are divided into three groups. Type I mGluRs, including mGluR1 and mGluR5, are the major targets of the Homer family and have been localized specifically around the periphery of PSD (Migaud et al., 1998; Thomas, 2002). AMPA-Rs have been shown originally to be clustered at excitatory synapses by GRIP (Dong et al., 1997). However, other reports also suggest that AMPA-Rs

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may interact with GRIP 2 without apparent clustering (Srivastava et al., 1998). It is remarkable that GRIP may interact with B-ephrin receptor tyrosine kinases, which have a pivotal role in the induction of LTP in hippocampal mossy fibers (Bruckner et al., 1999; Contractor et al., 2002).

E. PSD Proteins and the Concept of Microdomain An emerging concept strongly correlated to PSD organization as a multifunctional structure implicated in synaptic plasticity regulation is the junctional signaling microdomain (Delmas and Brown, 2002). Several lines of evidence suggest that the plasma membrane is specialized locally, both structurally and functionally, into distinct microdomains and subdomains. The suggested role of the microdomains is to ensure a highly specific and selective downstream signaling, avoiding interactions between molecules whose activation would introduce noise or contrasting eVects in the downstream propagation of the transduction signal. The PSD may have a significant impact in the organization of microdomains, as key molecules of the PSD are involved in signal transduction and scaVolding/ adaptor proteins are the elements that physically link, in a cooperative manner, diVerent intracytoplasmic ‘‘transducers.’’ The functional importance of PSD proteins in signaling microdomains architecture can be appreciated especially considering the role of Homer proteins (both the constitutive and the inducible isoforms, see later) in the spatial and temporal Ca2þ-regulated modulation of signal transduction. Homer constitutive proteins may interact through the EVH1/WH1 (EVH1, Ena/Vasp homology 1; WH1, Wasp homology 1) domain with type I mGluRs (whose activation may modulate the cytosolic calcium concentration) and IP3Rs (inositol 1,4,5-triphospate receptors). Moreover, the ryanodin receptor type I (RyR1) binds a proline-rich sequence of Homer (Shiraishi et al., 1999; Sun et al., 1998; Xiao et al., 1998). In this way, Homer acts as a scaVolding element that links together major molecules involved in the regulation of cytosolic calcium (Serge et al., 2002). In summary, junctional signaling microdomains may represent a highly eVective strategy developed to ensure specificity, speed, and selectivity in the propagation of signal transduction.

F. PSD Proteins and Receptor Diffusion A relevant role of postsynaptic density proteins with scaVolding and association function has been recognized also in receptor diVusion that, together with endocytosis and exocytosis, is a crucial mechanism regulating the movement of receptors in the postsynaptic membrane during plasticity (Blanpied et al., 2002;

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Tovar and Westbrook, 2002). It has been only recently that the role of lateral diVusion in setting the number of receptors functionally available for neurotransmitter binding has been fully appreciated. Both the disappearance of the receptors from the cell surface and the dispersal of receptors from the synaptic to extrasynaptic membrane may be responsible for an apparent net loss of receptors from a postsynaptic site. Receptor diVusion can be viewed as a lateral movement of receptors in the plane of the membrane mainly between synaptic and extrasynaptic pools (Choquet and Triller, 2003). An example of the relevance of postsynaptic density proteins and their complex interactions in receptor diVusion comes from the stargazin–PSD-95/SAP90 interaction in synaptic AMPA-R number regulation. AMPA receptors are localized to synapses by direct binding of the first two PDZ domain of PSD-95/SAP90 to the AMPA-R-associated protein, Stargazin. Increased levels of PSD-95 in hippocampal slice cultures recruit new AMPA-Rs to synapses, but this does not change the number of AMPA-Rs expressed to the membrane surface. Overexpression of Stargazin increases the level of extrasynaptic AMPA-Rs. Removing the PDZ-binding site of Stargazin, by deleting the last four amino acids at its C terminus, causes a reduction of its clustering and of AMPA excitatory postsynaptic currents (EPSC). Taken together, these findings point to a mechanism of AMPA-R transport between synaptic and extrasynaptic membrane, regulated by Stargazin binding to synaptic PSD-95/SAP90, trapping AMPA-Rs (Schnell et al., 2002). Fast movements and periods of receptor immobility have been described on the surface of neurites. The relative time spent in each state may be modulated by scaVolding protein expression. It is important to stress that when receptors are clustered by PSD proteins, they are not completely immobilized but may move within the cluster. In summary, many lines of evidence suggest an involvement of scaVolding and adaptor proteins in the regulation of receptor lateral diVusion.

III. PSD-95/SAP90

A. Structure PSD-95/SAP90 is an 80-kDa protein that migrates at 95 kDa on SDS–polyacrylamide gels and is a close homolog of the Drosophila protein Discs-large (Dlg) (Cho et al. 1992). PSD-95/SAP90 is a MAGUK protein that contains a catalytically inactive guanylate kinase-like (GK) domain in the carboxyl-terminal region, an Src homology 3 (SH3) domain in the central zone, and three PDZ (PDZ1, 2, 3) domains at the amino-terminal end. The GK domain binds GKAP directly, a

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Fig. 1. Domain organization and protein-binding partners of PSD-95/SAP90.

major constituent of PSD able to interact with Shanks/ProSAPs (see later) and therefore is important for protein–protein interaction (Kim et al., 1997) (Fig. 1). The GK and SH3 domains probably make a module responsible for the oligomerization of MAGUK scaVolding proteins. For example, several diVerent proteins, such as protein 4.1 and calmodulin, binding to the hinge region of the SH3 domain could subvert the intramolecular assembly of the SH3 and support intermolecular assembly (Lue et al., 1994; Masuko et al., 1999; McGee et al., 2001). The PDZ (PSD-95/Dlg/ZO-1) domains are modular domains of about 90 amino acids that bind specific sequences at the C termini portion of target proteins. Specifically, PDZ1 and PDZ2 can bind Shaker-type Kþ channels (Kv1.4), NMDA-R NR2 subunits, and Stargazin, a protein connecting PSD-95/SAP90 with AMPA-Rs (Chen et al., 2000; Kim et al., 1995; Kornau et al., 1995; Schnell et al., 2002); PDZ3 binds neuroligins (neuronal cell adhesion molecules) and CRIPT (a cysteine-rich interactor of PDZ3), a microtubule-associated protein (Irie et al., 1997; Niethammer et al., 1998). Furthermore, PDZ2 is able to bind neuronal nitric oxic synthase (nNOS) through a PDZ–PDZ interaction, establishing a crucial functional connection (Brenman et al., 1996a). Finally, SynGAP, a Ras-GTPase-activating protein, interacts with all three PDZ domains of PSD-95/SAP90 (Kim et al., 1998). Long et al. (2003) proposed that PDZ1 and PDZ2 behave in tandem, PDZ12, promoting the dimerization of receptors and ion channels or stabilizing the receptor dimer. B. Functions 1. PSD-95/SAP90 and Assembly of PSD The first steps of PSD-95/SAP90 clustering function are its multimerization ability through N-terminal domains and its anchorage property to the plasma membrane by palmitoylation of two N-terminal cysteines (Hsueh et al., 1997;

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Topinka and Bredt, 1998). Palmitoylation is crucial for molecule clustering and for the synaptic localization of PSD-95/SAP90 (Craven et al., 1999). This process in heterologous cells and primary hippocampal cultures may be prevented by conjugation with 2-bromopalmitate, resulting in dispersed synaptic clusters of PSD95/SAP90, end in a decreased number of synaptic AMPA-Rs (El-Husseini Ael et al., 2002). This finding supports the hypothesis that, through an interaction with Stargazin, AMPA-Rs localized into intracellular compartments are driven to the cell surface. These AMPA-Rs/Stargazin complexes are kept at the synapse by interaction with PSD-95/SAP90 (Schnell et al., 2002). PSD-95/SAP90 seems therefore a pivotal modulator of AMPA-R targeting. PSD-95/SAP90 also interacts with subunits of the kainate receptors. The KA2 subunit of the kainate receptor interacts with both SH3 and GK domains, whereas the GluR6 subunit binds specifically to the PDZ1 domain. This interaction leads to clustering and incomplete kainate receptor desensitization (Garcia et al., 1998). In addition, PSD-95/SAP90 cooperates with Kv1.4. When PSD-95/SAP90 is expressed heterologously together with Kv1.4, the expressed proteins become colocalized in plaque-like clusters whereas when the Kv1.4 channel and PSD-95/ SAP90 are expressed individually, the proteins are localized diVusely throughout the cellular membranes or the cytosol (Kim et al., 1995). PDZ2 seems to be a key domain for Kv1.4 clustering (Imamura et al., 2002). These findings are supported by evidence that the genetic disruption of D1g (see earlier discussion) causes great changes in the morphology and physiology of synapses and interferes with synaptic localization of Kv1.4. (Budnik, 1996; Tejedor et al., 1997). However, work by Rasband et al. (2002) denies the clustering ability of PSD-95/SAP90. Indeed, they have found, at juxtaparanodal regions adjacent to the node of Ranvier, that Kv1 clustering is normal in a mutant mouse lacking juxtaparanodal PSD-95/SAP90 (Rasband et al., 2002). Further studies are needed in order to clarify this issue. PSD-95/SAP90 synaptic interaction with NMDA-Rs is not yet completely defined. In mutant mice lacking PSD-95/SAP90, the localization of NMDA-R is normal and, according to this finding, mice expressing the NR2 subunit in a C-terminally truncated form show no change in the synaptic localization of NMDA-R (Migaud et al., 1998; Sprengel et al., 1998). Nevertheless, the NR2A subunit in a C-terminally truncated form impairs synaptic but not extrasynaptic localization of NMDA-Rs; moreover, during synapse development, PSD-95/ SAP90 clusters before NMDA-R apparently form a scaVold to which the receptors later attach (Rao et al., 1998; Sanes and Lichtman, 1999; Steigerwald et al., 2000). 2. PSD-95/SAP90 and Synaptic Plasticity More than 100 molecules have been associated with synaptic plasticity, namely with LTP and long-term depression (LTD) (Sanes and Lichtman, 1999), nevertheless the role of PSD-95/SAP90 in the development of these events

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may be crucial. Migaud et al. (1998) demonstrated that mutant mice lacking PSD95/SAP90 have an enhancement of LTP and a learning impairment. Mutant mice showed a significantly larger potentiation of synaptic transmission so that various stimulation frequencies (1, 5, 10, 20, 100 Hz) induce LTP. Furthermore, the analysis of spatial learning by means of the water maze test demonstrated that mutant mice have a marked inability to learn the position of the hidden platform. This enhanced LTP seems unrelated to the SynGAP–MAPK pathway (Grant and O’Dell, 2001; Komiyama et al., 2002). Moreover, PSD-95/SAP90 connects NMDA-Rs with nNOS, enzyme well known to be involved in LTP and LTD induction (Christopherson et al., 1999; Wu et al., 1997). This association may modulate nNOS function. Suppressing the expression of PSD-95/SAP90 attenuates nitric oxide neurotoxicity (Sattler et al., 1999). PSD-95/SAP90 also promotes nNOS phosphorylation at Ser847 induced by CaMKII, which reduces enzyme catalytic activity (Komeima et al., 2000; Watanabe et al., 2003). PSD-95/SAP90 functionally modulates NMDA-Rs and its overexpression increases AMPAR-mediated synaptic transmission (Beique and Andrade, 2003; Yamada et al., 2002). This last finding is related to a previous report demonstrating that PSD95/SAP90 overexpression increases synaptic AMPA-R levels, miniature excitatory postsynaptic current (mEPSCs) amplitude, and the size and number of dendridic spines (El-Husseini et al., 2000). According to these results, PSD-95/ SAP90 reduced expression seems responsible for hippocampal neuronal death through CaMKII transduction pathway activation (Gardoni et al., 2002).

C. PSD-95/SAP90 and Neuropsychiatric Disorders Evidence suggests that PSD-95/SAP90 may be involved in the pathophysiology of some neuropsychiatric disorders, such as schizophrenia, ischemia, and Huntington’s disease (HD). The glutamatergic system is involved in the pathophysiology of psychosis. Indeed, NMDA-R hypofunction has been proposed as a model of schizophrenia (Olney and Farber, 1995). NMDA noncompetitive receptor antagonists, such as ketamine and phencyclidine (PCP), induce a schizophrenia-like psychotic state in normal subjects that closely mimics both positive and negative symptoms of the disorder and that is suppressed by treatment with clozapine but not with haloperidol ( Javitt and Zukin, 1991; Krystal et al., 1994, 1999; Malhotra et al., 1997). Moreover, ketamine and PCP may exacerbate psychotic symptoms in schizophrenic patients (Lahti et al., 1995). Two studies investigated PSD-95/SAP90 mRNA expression in diVerent brain areas of schizophrenic subjects. In the first one, in situ hybridization histochemistry (ISHH) showed a significant PSD-95/SAP90 gene expression decrease in Broadmann’s area 9 of the prefrontal cortex but not in the hippocampus of

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schizophrenic patients compared to normal controls (Ohnuma et al., 2000). In the second one, Dracheva et al. (2001) compared PSD-95/SAP90 expression in patients with schizophrenia, patients with Alzheimer’s disease, and normal controls. The level of PSD-95/SAP90 mRNA expression in the occipital cortex (Broadmann’s area 17) of schizophrenic subjects was significantly higher compared to normal subjects, where no significant changes were observed in the dorsolateral prefrontal cortex (Broadmann’s area 46). PSD-95/SAP90 gene expression was unchanged in the brains of the Alzheimer’s disease group (Dracheva et al., 2001). In animal models, NMDA-R blockade induced by the administration of MK801, an uncompetitive NMDA-R antagonist, produces a significant increase of PSD-95/SAP90 mRNA levels in parietal, entorhinal, temporal, and perirhinal rat corticies (Linden et al., 2001). Moreover, PSD-95/SAP90 gene expression is unmodified after the acute administration of antipsychotics drugs such as haloperidol and olanzapine in several cortical and subcortical regions, including the prefrontal cortex (cG30 area) caudate-putamen, and accumbens (de Bartolomeis et al., 2002). These findings, taken together, suggest an involvement of PSD-95/ SAP90 in schizophrenia, but other studies are needed to better understand the glutamatergic transmission role in the pathophysiology of psychosis. The first evidence of a PSD-95/SAP90 implication in brain ischemia came from the studies of Hu et al. (1998) and Takagi et al. (2000). These authors demonstrated that an ischemic challenge produces a slight decrease of PSD-95/ SAP90 expression (Hu et al. 1998), a decreased association of PSD-95/SAP90 with NR2A and NR2B NMDA-R subunits, and a reduction in the size of protein complexes containing PSD-95/SAP90 (Takagi et al., 2000). A pivotal report by Aarts et al. (2002) demonstrated that perturbing in vivo the interaction between PSD-95/SAP90 and NMDA-Rs reduces the ischemic brain damage induced in rats by transient middle cerebral artery occlusion (MCAO) by the intraluminar suture method. The administration of peptides that bind PSD-95/SAP90 PDZ2 domains, dissociating the NMDA-R–PSD-95/SAP90 complex, decreases the total cerebral infarction volume and improves motor deficits (Aarts et al., 2002). This finding may suggest new strategies for treating stroke. Huntington’s disease is a dominant inherited neurodegenerative disorder characterized by choreiform movement, psychiatric disturbances, and cognitive decline (Martin and Gusella, 1986). HD is caused by a polyglutamine expansion of huntingtin, the protein codified by the HD gene (The Huntington’s Disease Collaborative Research Group, 1993). Normal huntingtin is enriched in dendrites and nerve terminals and is associated with microtubule complexes and synaptic vescicles. It binds the SH3 domain of PSD-95/SAP90 through its N-terminal proline region, while polyglutamine expansion inhibits the interaction between huntingtin and PSD-95/SAP90. Furthermore, in HD patients the huntingtin association with PSD-95/SAP90 is about 80% lower compared to normal subjects

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(Sun et al., 2001). These data suggest that normal huntingtin sequesters PSD-95/ SAP90, avoiding neuronal toxicity, whereas polyglutamine-expanded huntingtin may sensitize and activate NMDA-Rs, causing neuronal toxicity. New findings support the view that PSD-95/SAP90 is also involved in neuropathic pain and in kainic acid-induced seizures (Garry et al., 2003; Wyneken et al., 2001).

IV. Shank/ProSAP Proteins

A. Structure Shank/ProSAP (SH3 domain and ankyrin repeat containing protein/ proline-rich synapse-associated protein) proteins are a family of three members: Shank1, Shank2, and Shank3. Shanks/ProSAPs are multidomain proteins containing, from N to C termini the following structures: an ANK (ankyrin) repeat region, a SH3 domain, a PZD domain, a proline-rich (PRO) region, a ppI (cortactin binding) domain, and a sterile  motif (SAM) domain (Fig. 2). The domain organization of Shank/ProSAP proteins appears to be regulated by alternative splicing, Shank1 lacks the ppI domain, and ANK seems to be absent in Shank2 (Boeckers et al., 1999; Lim et al., 1999). In rat brain, Shank/ProSAP mRNAs are expressed diVerentially in several brain areas, namely Shank1 is abundant in the cortex, hippocampus, and amygdala; Shank2 and Shank3 show a similar distribution in the cortex and hippocampus, but in the cerebellum, Shank2 is found in Purkinje cells, whereas Shank3 is only expressed in the granule cell layer (Sheng and Kim, 2000). The ANK repeat region of Shank1 and Shank3 binds -fodrin, a multidomain protein containing 22 spectrine repeats, one SH3 domain, and two EF hand calcium-binding motifs (Carlin et al., 1983). -Fodrin is a cytoskeletal protein

Fig. 2. Domain organization and protein-binding partners of Shank/ProSAP proteins.

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interacting with actine and calmodulin, expressed in the neuronal dendritic compartment mainly in the dendridic spines and in PSD (Glenney et al., 1983). The ANK repeat region also interacts with the C-terminal zone of Sharpin, a PSD component able to homomultimerize through the N-terminal extreme (Lim et al., 2001). Therefore, Sharpin may cross-link multiple Shank proteins. The SH3 domain interacting partners are to date unclear, indeed Sheng and Kim (2000) reported, in GST-pulldown experiments, that the SH3 domain binds to GRIP, but no more studies have been published to support this finding. The PDZ domain binds GKAP, somatostatin receptor type 2 (SSTR2), the calcium-independent latrotoxin receptor (IRL), and PIX, a guanine nucleotide exchange factor for Rac1 and Cdc42 small GTPases (Boeckers et al., 1999; Kreienkamp et al., 2000; Park et al., 2003; Zitzer et al., 1999a,b). The interaction with GKAP links, through PSD-95/SAP90, the NMDA-R macromolecular complex with Shanks/ProSAPs. The PRO region is more than 1000 residues long and is rich in proline and serine. Three proteins shown to bind to the PRO region are Homer, dynamin-2, and IRSp53 (insulin receptor tyrosine kinase substrate protein). Homer (see later) is a scaVolding molecule linking type I mGluRs with releasable Ca2þ intracellular pools (Tu et al., 1998, 1999). Dynamin-2 is a member of endocytic machinery, interacting with a short serine-rich sequence within the PRO region (Okamoto et al., 2001). IRSp53 is a signaling molecule that acts downstream of small G-proteins such as cdc42 and rac (Bockmann et al., 2002; Soltau et al., 2002). The ppI domain is a prolin-rich cluster within the PRO region that mediates the interaction with cortactin, an F-acting-binding protein enriched in cell matrix contact sites, in lamellipodia of cultured cells, and in growth cones of cultured neurons (Du et al., 1998; Wu and Parsons, 1993). Furthermore, cortactin translocates to the cell periphery by means of rac1 and reallocates to synapses after glutamate stimulation (Naisbitt et al., 1999; Weed et al., 1998). Therefore, cortactin may play a key role in the cytoskeletal organization produced by intracellular and extracellular stimuli. The SAM domain of Shanks/ProSAPs interacts with itself in vitro and may be involved in a tail-to-tail multimerization of Shank proteins (Naisbitt et al., 1999).

B. Functions Although the Shank/ProSAP structure is well characterized, to date only two reports can help us understand Shank/ProSAP functions. Sala et al. (2001) showed that Shank1 overexpression, in transfected primary hippocampal neurons, stimulates the maturation and growth of dendritic spines. Namely, Shank1 expression during the first week, when endogenous Shank1 is expressed at low levels, accelerates the development of mushroom spines; moreover, during

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the second week, when endogenous Shank1 is increased, Shank1 overexpression promotes spine head enlargement. Furthermore, dominant-negative Shank1 mutants cause a reduction in spine density. Intriguingly, Shank1 postsynaptic overexpression seems to enhance a presynaptic function (Sala et al., 2001). Park et al. (2003) demonstrated that overexpression of Shank1B in cultured hippocampal neurons promotes recruitment of PIX and PAK (p21-activated kinase) in dendritic spines. PIX is a guanine nucleotide exchange factor for the Rac1 and Cdc42 small GTPases; PAK is a family of Rac/Cdc42-activated serine/threonine kinases that, through PIX, interacts with Rac1/Cdc42 (Bagrodia and Cerione, 1999; Manser et al., 1998). PIX activates Rac1 and Cdc42 small GTPases involved in neurite initiation, growth, guidance, branching, polarity, and synapse formation (Luo, 2000). These findings suggest that Shank may regulate spine dynamics through the synaptic accumulation of PIX IX and local activation of the Rac1-PAK signaling pathway. C. Shank3 and the 22q13.3 Deletion Syndrome The 22q13.3 deletion syndrome is a neurogenetic syndrome characterized by severe expressive language delay, mild mental retardation, and facial dysmorphisms (Nesslinger et al., 1994; Prasad et al., 2000). The first indication of a Shank3 gene involvement in 22q13.3 deletion syndrome came from Wong et al. (1997). In a child with clinical signs of 22q13.3 deletion syndrome (severely delayed expressive language, mild mental retardation, hypotonia, dolichocephaly, epicanthic folds, bulbous nose, and lax joints), Bonaglia et al. (2001) showed a balanced translocation between chromosomes 12 and 22, causing a disruption of the Shank3 gene at exon 21. This result suggests a Shank3 association with this syndrome (Bonaglia et al., 2001). V. SAP97

A. Structure Synapse-associated protein 97 kDa (SAP97), together with PSD-95/SAP90, SAP102, and PSD-93/chapsyn-110, is a member of the MAGUK protein family (Muller et al. 1995). SAP97 is a multidomain protein, characterized from the C to N-terminal region by a GK domain, a SH3 domain, and three PDZ domains. Three further regions can be recognized: the HOOK/U5 region (protein sequences situated between the GK and the SH3 domain), the S97N region (protein sequences N-terminal to PDZ1), and the MRE (MAGUK recruitment) domain (Fig. 3) (Karnak et al., 2002; Lee et al., 2002; Wu et al., 2002). The

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Fig. 3. Domain organization and protein-binding partners of SAP97. The SAP97 interaction with the NR2A subunit of the NMDA receptor is not shown.

S97N region and the MRE domain may coincide, but because this issue is not yet clear, they will be dealt with separately. The GK domain binds GKAP; interestingly, this association is inhibited by intramolecular interactions of the SH3 domain and HOOK/U5 region with the GK domain and is enhanced by the S97N region binding with the SH3 domain and HOOK/U5 region ( Wu et al., 2000). The GK and SH3 domain of SAP97 mediate binding to AKAP79/150 (A-kinase anchoring protein 79 kDa in humans and 150 kDa in rodents), a protein binding the regulatory subunits of protein kinase A (PKA) and targeting PKA to various subcellular compartments (Colledge et al., 2000; Colledge and Scott, 1999). The HOOK/U5 region interacts with calmodulin, the principal mediator of calcium-dependent signaling, with low to intermediate aYnity in a calcium-dependent manner (Paarmann et al., 2002). The PDZ1 domain binds the GluR6 subunits of kainate receptors weakly and has been proposed to interact with GluR1 subunits of AMPA-Rs (Mehta et al., 2001). The PDZ2 domain interacts with GluR1 subunits of AMPA-Rs (Leonard et al., 1998). This association is specific; indeed, GluR1 does not bind PDZ domains of other MAGUKs and is dependent on a GluR1 SSG sequence located outside the PDZ-binding motif (Cai et al., 2002). The PDZ2 domain is also able to bind the inward rectifier potassium channels Kir2.2 and Kir3.2c (Hibino et al., 2000; Leonoudakis et al., 2001). The PDZ3 domain is in charge of binding with tumor necrosis factor  converting enzyme (TACE), an enzyme responsible for ectodomain shedding of TNF- (Peiretti et al., 2003). Finally, Bassand et al. (1999) showed that the C-terminal tail of NR2A interacts strongly with portions of SAP97 encompassing the three PDZ domains, suggesting a SAP97 interaction with NMDA-R (Bassand et al., 1999). The MRE domain binds DLG3 (discs large 3), DLG2 (discs large 2), and calcium/calmodulin-dependent serine protein kinase (CASK), three members of MAGUK family proteins (Karnak et al., 2002). The S97N region binds myosin VI, a minus

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end-directed actin-dependent motor protein, able to move the particles/vesicles toward the minus or pointed end of actin filaments (Cho et al., 1992; Wells et al., 1999; Wu et al., 2002). Whereas other MAGUKs were found only in neuronal cells, SAP97 is also located in non neuronal tissues such as epithelial/endothelial, mesenchymal, and hematopoietic cells (Brenman et al., 1996b; Caruana and Bernstein, 2001; Cho et al., 1992). Furthermore, it has been discovered only recently that SAP97 is both presynaptic and postsynaptic and that it is enriched in PSD ( ValtschanoV et al., 2000). SAP97 is found in diVerent brain areas such as the cerebellum and forebrain, but is especially expressed in the pyramidal layers of the hippocampus, where SAP97 is located in apical dendrites through the stratum radiatum and stratum lucidum in the CA1 and CA3 areas, respectively (Bassand et al., 1999).

B. Functions SAP97 is involved in AMPA-R targeting. The interaction between SAP97 and GluR1 subunits occurs early in the secretory pathway, whereas the receptors are in the endoplasmic reticulum or cis-Golgi (ER-CG) (Sans et al., 2001). SAP97 is enriched in the PSD, but is also abundant in the cytoplasm and interacts with intracellular membranes. This peculiar intracellular localization is consistent with a role in the organization of membrane proteins and seems not related to GK and SH3 domain truncation (Klocker et al., 2002). Moreover, few synaptic AMPA-Rs bind SAP97. These findings may suggest that SAP97 carries GluR1 from ER-CG to the plasma membrane and, once there, dissociates from the receptor complex (Sans et al., 2001). Considering that SAP97 binds myosin VI (see earlier discussion), a suggestive hypothesis is that SAP97 serves as a molecular link between GluR1 and myosin VI during the translocation of AMPA-Rs to and from the postsynaptic membrane ( Wu et al., 2002). SAP97 is probably involved in hippocampal synaptic plasticity. The phosphorylation state of the GluR1 subunit is most likely associated with AMPA-R current intensity; indeed the phosphorylation of Ser845 on GluR1 enhances native and recombinant AMPA-R currents, whereas Ser845 is dephosphorylated during LTD (Banke et al., 2000; Lee et al., 2000). SAP97 binds AKAP79/150 (see earlier discussion), a multivalent anchoring protein associated with PKA and PP2B ( protein phosphatase 2B). PKA and PP2B are, respectively, in charge of GluR1 phosphorylation and dephosphorylation (Colledge et al., 2000; Tavalin et al., 2002). Taken together, these results demonstrate that SAP97 is a member of a complex involved in the modulation of GluR1 receptor currents. Moreover, SAP97 may regulate Kir3.2c function. Kir3.2c is a neuronal inwardly rectifying Kþ channel activated directly by G-proteins, is coupled to

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several inhibitory receptors, is involved in the generation of slow inhibitory postsynaptic potentials, and is localized at the postsynaptic membrane in dopaminergic neurons (Inanobe et al., 1999; Luscher et al., 1997; Yamada et al., 1998). Hibino et al. (2000) showed that SAP97 induces the sensitization of Kir3.2c to G-protein stimulation, and the GK domain seems to play a major role in this function (Hibino et al., 2000).

C. SAP97 and Schizophrenia Toyooka et al. (2002) showed an association of SAP97 with schizophrenia. The authors have investigated the expression of several PDZ proteins, including PSD-95/SAP90, SAP97, PSD-93/chapsyn-110, GRIP1, and SAP102 in postmortem human brain tissue of schizophrenic patients by means of Western blot analysis. The regions analyzed were the dorsolateral prefrontal cortex (Broadmann’s area 46), the occipital cortex (Broadmann’s area 17), CA regions, and the dentate gyrus of the hippocampus. SAP97 levels were decreased significantly in the dorsolateral prefrontal cortex of schizophrenics, where GluR1 protein expression was also reduced. This finding suggests that SAP97 reduction may contribute to glutamatergic dysfunction in the dorsolateral prefrontal cortex. Intriguingly, levels of other PDZ proteins (PSD-95/SAP90, chapsyn-110, GRIP1) were found unmodified in the prefrontal cortex of schizophrenic subjects versus the control group (Toyooka et al., 2002). In an animal model, uncompetitive NMDA-R antagonists modify SAP97 mRNA expression. Indeed, MK-801 and phencyclidine administration increases SAP97 levels in the entorhinal cortex, whereas in superficial layers of the parietal cortex, treatment with MK-801 decreases SAP97 mRNA expression (Linden et al., 2001). These findings represent further clues that the glutamatergic system is involved in the pathophysiology of psychosis.

VI. Homer Proteins

A. Structure Homer is a family of proteins that includes the following members: Homer 1b/c, 2a/b, 3, and Homer 1a. Homer 1b/c, 2a/b, and 3, called coiled-coil (CC), are expressed constitutively, whereas Homer 1a is inducible (Xiao et al., 1998). Genes coding for Homers are located, in humans, on chromosome 5 (Homer1), chromosome 15 (Homer2), and chromosome 19 (Homer3) (Xiao et al., 1998). Both constitutive and inducible isoforms are characterized by an

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Fig. 4. Domain organization and protein-binding partners of Homer proteins.

EVH1/WH1 domain at the amino-terminal region, while only CC isoforms have a coiled-coil domain at the carboxyl-terminal extreme (Fig. 4). The EVH1/WH1 domain is about 110 amino acids long and, binding to PPxxF (single-letter amino acid code used, where ‘‘x’’ is any amino acid) motifs, mediates association with type I mGluRs, IP3Rs, RyR1, Shank/ProSAP proteins and dynamin 3 (Dyn3) (Brakeman et al., 1997; Feng et al., 2002; Gray et al., 2003; Tu et al., 1998, 1999). Type I mGluRs include mGluR1 and mGluR5, which are coupled to phospholipase C and activate phosphoinositide hydrolysis to produce IP3 and diacylglycerol (Nakanishi, 1994). IP3Rs and RyR1 play a central role in releasable Caþ2 intracellular pool modulation. Dyn3 is a mechanoenzyme localized within the PSD involved in the maintenance of dendritic morphology by regulating the outgrowth of dendritic protrusions and the morphogenesis of dendritic spines (Gray et al., 2003). The CC domain, about 200 amino acids long, is required for self-multimerization and contains typical leucine zipper motifs mediating homotypic interactions (Tadokoro et al., 1999; Xiao et al., 1998). Homer 1a lacks the CC region and therefore is unable to multimerize. The open reading frame spreads over 10 exons. Exon 1 codes for the 50 untranslated region (UTR), exons 2–5 code for the N-terminal EVH1 domain, and exons 6–10 code for the C-terminal CC motif and the 30 UTR. A substantial number of modulation elements have been recognized in the Homer1 gene promoter region, including SP1, AP1, GATA, E-box, and CRE (Bottai et al., 2002). Homer 1a presents a unique and highly conserved C-terminal tail of 11 residues. This 11 residue tail is coded by a 33 nucleotide sequence positioned into the

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intron between exon 5 and exon 6, and an alternative splicing, most likely triggered by synaptic activity (see later), allows the ending of the Homer 1a transcripts within intron 5 (Bottai et al., 2002). This mechanism extends the exon 5 sequence and generates an mRNA construct constituted by EVH1 domain coding exons but not by exons coding for the CC region. A similar transcription mechanism is also present in Ania-3, a less investigated activity-induced splice variant of Homer 1 that shows great similarity with Homer 1a. Further, Homer 1a mRNA contains several AUUUA repeats at 30 UTR. The AUUUA repeats are responsible for destabilizing the interaction with the translational machinery at the ribosomal level. The functional consequence of this organization of gene structure is a fast decay of the mRNA transduction (Bottai et al., 2002). The AUUUA repeats are common to other IEGs, while they lack in the Homer 1b/c mRNA (Soloviev et al., 2000; Xiao et al., 1998). However, compared to other early genes, e.g., c-fos, the mRNA for Homer 1A lasts relatively longer after its induction by synaptic activity. The fast and dramatic increase of Homer1a mRNA after stimuli is not paralled by an increase of similar magnitude in the Homer1a protein level (Kato et al., 1998). A possible explanation is a rapid degradation pattern of the protein due to the presence of a PEST sequence in the Homer1 and perhaps Homer2 C-terminal regions (Soloviev, 2000). PESTs are amino acid sequences acting as molecular markers. Indeed, proteins displaying PEST sequences in their primary structure are degraded by an obiquitinemediated system. Moreover, PEST sequences have been found particularly in proteins with a high turnover. However, the fast degradation pattern is typical of Homer 1a but not Homer 1b, Homer 2, or Homer 3 proteins (Ageta et al., 2001), whereas the PEST sequence is shared by almost all Homer isoforms. Because Homer 1a mutants lacking the 11 residue C-terminal tail are not degraded rapidly, it seems likely that the Homer 1a unique 11 residue tail might promote its fast degradation pattern (Ageta et al., 2001). It is remarkable that Homer proteins, as well as SPA97, have been identified not only in the nervous system, but also in various peripheral tissues, including cardiac and skeletal muscles (Sandona et al., 2000). Saito et al. (2002) found a novel Homer 1 isoform (Homer 1d) from the cardiac cDNA library identical to Homer 1b except for a unique N-terminal region.

B. Functions TraYcking of type I mGluRs is probably the most extensively investigated function of Homer proteins. Type I mGluR surface expression has been studied in diVerent heterologous expression systems. In HeLa cells, Homer 1b was found to trap mGluR1a and mGluR5 in the endoplasmic reticulum, causing a reduction of receptors at the plasma membrane. Moreover, mGluR5 point mutations

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interrupting the association with Homer inhibit this eVect and Homer 1a expression does not modify mGluR5 surface expression (Roche et al., 1999). In HEK-293 cells, both Homer 1c and Homer 1a induce plasma membrane surface clustering of mGluR1a (Ciruela et al., 1999, 2000). In contrast, Coutinho et al. (2001) showed, also in HEK-293 cells, that neither Homer 1a nor Homer 1c had any marked eVect on mGluR1a distribution, whereas coexpression of mGluR5 with Homer 1c resulted in reduced receptor surface localization and receptor intracellular cluster formation (Coutinho et al., 2001). In COS-7 cells, Homer 1c causes mGluR1a clustering but has no eVect on membrane targeting of mGluR1a (Tadokoro et al., 1999). Ango et al. (2002) investigated the role of Homer 1a and Homer 1b in the traYcking of mGluR5 in primary cultures of mouse cerebellar granule cells. In this paradigm, Homer 1b produces intracellular clustering of mGluR5 at synaptic sites, whereas Homer 1a inhibits the intracellular retention of receptors, allowing cell surface localization of mGluR5 at synaptic sites. Furthermore, Homer 1a increases the latency and the amplitude of mGluR5-mediated Ca2þ responses (Ango et al., 2002). Homer proteins can also regulate mGluR localization in specific neuronal compartments. Indeed, in cultured cerebellar granule cells, in the absence of Homer 1, mGluR5 are localized in the soma, whereas Homer 1b distributes mGluR5 at the dendritic synaptic sites and Homer 1a causes mGluR5 translocation to both dendrites and axons (Ango et al., 2000). Moreover, in cultured cortical neurons, Homer 1c increases the transport of mGluR1a to the dendrites (Ciruela et al., 2000). In addition, Homer proteins are involved in axon pathfinding in vivo. Indeed, in Xenopus optic tectal neurons, time-lapse imaging shows that interfering with Homer 1b/c causes axon pathfinding errors (Foa et al., 2001). Homer proteins connect type I mGluRs with releasable Caþ2 intracellular pools through IP3Rs and RyR1. This interaction links type I mGluRs with their downstream eVectors. Homer 1a expression, in transfected Purkinje cells, causes an amplitude reduction and a latency increase of mGluR-evoked Caþ2 responses, as compared with Homer 1b transfection (Tu et al., 1998). Furthermore, it is remarkable that both Homer 1b and Homer 1a might bind RyR1 and similarly enhance their responses to physiological and pharmacological stimuli such as Ca2þ, depolarization, and caVeine (Feng et al., 2002). Homer proteins modulate the coupling of type I mGluRs to N-type calcium and M-type potassium channels. Indeed, Homer CC may occlude signaling from type I mGluRs to G-proteins regulating N-type calcium and M-type potassium channels, whereas Homer 1a may revert this eVect (Kammermeier et al., 2000). Homer proteins may also modulate type I mGluR activation. Indeed, a pivotal paper of Ango et al. (2001) demonstrated, in cultured cerebellar granule cells, that Homer 3 knock down and Homer 1a induction produce an agonistindependent mGluR1a activation. Taken together, these findings suggest a model in which Homer 1a behaves as ‘‘dominant negative,’’ promoting disassembly of

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the type I mGluR signaling complex, modulating glutamatergic synaptic activity directly. Homers interaction with Shank proteins is involved in the regulation of dendritic spine morphology, namely the ability of Shank1 to induce spine enlargement (see earlier discussion) seems related to calcium release from intracellular stores. This release may be due to the synaptic recruitment of IP3Rs by Homers (Sala et al. 2001).

C. Homer 1a Induction and Implication for Neuropsychiatric Disorders Homer 1a is an immediate early gene (IEG), and several stimuli may induce its transient transcription. In a pivotal paper, Brakeman et al. (1997) demonstrated that Homer 1a mRNA upregulation in the hippocampus after electroconvulsive seizure and synaptic stimulus in the cortex by visual experience and in striatum by cocaine administration produces LTP (Brakeman et al., 1997). These data led some researchers to explore Homer 1a expression in diVerent experimental paradigms. In E1 mice, an animal model for human epilepsy, within 1 h from a tonic– clonic seizure, Homer 1a mRNA increases in the granule cell layer of dentate gyrus (DG) and a weaker signal occurs in pyramidal cells of the hippocampus and cortex. No significant modification was observed 8 h after seizure, suggesting a Homer 1a transient increase due to the tonic–clonic seizure (Morioka et al., 2001). In the rat kindling model of temporal lobe epilepsy, Potschka et al. (2002) demonstrated, by means of massively parallel signature sequencing and quantitative RT-PCR, a Homer 1a induction in the hippocampus of kindled rats. Furthermore, kindling of transgenic mice overexpressing Homer 1a was retarded significantly compared to wild-type mice. These results suggest that Homer 1a may have anticonvulsant and antiepileptogenic eVects (Potschka et al., 2002). In rats, phencyclidine administration (see earlier discussion) has been reported to acutely induce Homer 1a mRNA in the prefrontal cortex and primary auditory cortex, and 24 h posttreatment, Homer 1a mRNA reduction in the retrosplenial cortex and dentate gyrus (Cochran et al., 2002). We also observed a Homer 1a increase in subcortical regions induced by subanesthetic and subconvulsant doses of ketamine, a dissociative anesthetic related structurally to PCP and with a complex receptor profile including a noncompetitive NMDAR blockade (A. de Bartolomeis et al., in preparation). Due to the role of NMDA hypofunction in several animal models of behavioral disorder, it may be intriguing to further explore the putative role of the Homer family in the pathophysiology of psychosis-like conditions. A Homer involvement in the mechanism of action of drugs used in the treatment of psychosis is suggested by Homer 1a induction after the acute administration of antipsychotics. Indeed, we have shown, by means of ISHH, that

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acute administration of antipsychotics with diVerent pharmacodynamic profiles modulates Homer 1a expression with a specific pattern. Namely, haloperidol induced an increase of Homer 1a mRNA in the caudate-putamen and in both core and shell of the nucleus accumbens, whereas olanzapine does so in the core of nucleus accumbens only (de Bartolomeis et al., 2002). Moreover, we have investigated Homer 1a gene expression after acute treatment with haloperidol or clozapine alone or with the coadministration of d-cycloserine, a glycine partial agonist acting at the glycine-binding site of NMDA-R. This paradigm reproduces in animals an augmentation strategy proposed for schizophrenia treatment (GoV et al., 1999a,b). In this pharmacologic isomorphism, we have observed that haloperidol produces a Homer 1a mRNA increase in the caudate-putamen and nucleus accumbens, whereas clozapine induces Homer 1a only in the accumbens. Intriguingly, Homer 1a induction after both haloperidol and clozapine was attenuated in the same brain areas after the adjunction of d-cycloserine (Polese et al., 2002). Taken together, these findings suggest that Homer 1a is induced diVerently by typical (haloperidol) or atypical (olanzapine, clozapine) antipsychotics and suggest a potential role for Homer as a molecular link between glutamatergic and dopaminergic transmission. Drug addiction is considered a disorder of synaptic plasticity taking place in specific brain areas known to be linked to reward, craving, and withdrawal. Evidence suggests Homer 1a involvement in opioid addiction. Indeed, Ania-3 mRNA, a splice variant of Homer 1 almost identical to Homer 1a, was found to be increased in the rat prefrontal cortex after chronic treatment with morphine. After administration of naloxone, an opioid antagonist precipitating withdrawal, Ania-3 mRNA levels were still elevated, providing a clue of long-lasting induction (Ammon et al., 2003). Even if the exploration of Homer expression and function in the central nervous system (CNS) by psychotropic compounds is only beginning, it is worth noting that chronic imipramine treatment does not modify Homer 1a protein levels in any brain region, underlining that only certain specific psychotropic drugs may modulate Homer 1a expression (Matrisciano et al., 2002). Memory and learning are key functions in behavioral adaptation and may represent a higher expression of a modulation of complex synaptic plasticity. Interaction with a novel environment may be considered a simple and relatively naturalistic example of memory and learning task. Using fluorescence in situ hybridization, Vazdarjanova et al. (2002) examined Homer 1a mRNA expression in a paradigm consisting in exploration of a novel environment for 5 min. Rats were sacrificed at 0, 8, 16, 25, or 35 min after exploration and confocal images were acquired for qualitative or quantitative analysis. Qualitative analysis has shown Homer 1a expression at 25 min in hippocampal CA1 and CA3 regions, and in the parietal cortex, these results were confirmed by quantitative analysis of the

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CA1 region demonstrating a significant increase of Homer 1a in the 25- and 35-min groups ( Vazdarjanova et al., 2002). These findings may provide evidence for a role of Homer 1a in learning and memory processes. Synaptic plasticity is believed to be aVected by light and circadian rhythms. Light induces a phase shift of activity rhythms only during a subjective night. Early gene activation in brain regions driving or related to circadian rhythms has been reported to be a neuronal marker of CNS pathways implicated in response to environmental light changes. The first studies on Homer activation showed that Homer mRNA expression is induced rapidly in rat suprachiasmatic nucleus by light during the subjective night, pointing to an involvement of this protein and perhaps of the glutamate metabotropic receptormediated signaling system in circadian rhythm-related synaptic changes (Park et al., 1997). More recently, this issue has been investigated further with the finding that Homer1a mRNA exhibits a diurnal variation in the rat suprachiasmatic nucleus in vivo with the highest level during the midsubjective day (Nielsen et al., 2002). These results, taken together, point to the potential contribution of Homer in synaptic changes that may be relevant for behavior and disorders of behavior linked to the abnormal regulation of circadian rhythms in mammals. Finally, an elegant piece of evidence that strongly links Homer gene regulation to the synaptic plasticity and adaptation has been reported by Bottai et al. (2002), who demonstrated a synaptic activity-induced conversion of the intronic to exonic sequence in Homer 1a expression. By means of fluorescent in situ hybridization, these authors showed that in mouse at a resting state of neuronal activity the entire Homer gene is transcribed constitutively to yield Homer 1b/c. After maximal electroconvulsive shock, a neuronal increase in the rate of transcription was detected, with most transcripts ending within the central codon (Bottai et al., 2002). In reviewing the role of Homer in synaptic plasticity disorders and its putative involvement in behavior, a report on Homer gene manipulation in Drosophila that may shed light on Homer function in unexpected animal behaviors needs to be considered (Diagana et al., 2002). A single gene encoding a protein homologous to the mammalian Homers has been identified (Kato et al., 1998; Xiao et al., 1998) and shown to be expressed in the CNS of Drosophila where it is localized in the endoplasmic reticular and targeted to dendritic spines. A mutant fly carrying a deletion in the Homer gene (Homer102), which removes the first two exons and half of the third exon, was generated. No gross abnormalities in brain morphology were observed in mutant flies compared to the wild type. However, mutation of Drosophila Homer disrupted the control of locomotor activity and was responsible for deficits in courtship conditioning (Diagana et al., 2002). Caution in translational neuroscience is always necessary, especially with behavioral findings in lower species. However, the role of the Homer signaling

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pathway in mating behavior is certainly fascinating and it would be interesting to explore its potential involvement in similar behavioral patterns in mammalians.

VII. Conclusive Remarks

Converging evidence suggests a pivotal role of PSD proteins in neurodevelopment, axon pathfinding, receptor number regulation and clustering, and maintenance of dendritic architecture, as well as in synaptic plasticity complex expression such as LTP. It may be conceivable that disruption of the normal structure and function of scaVolding, adaptor, or kinase proteins at PSD may strongly impact synaptic plasticity. Animal models and pharmacologic isomorphisms, as well as genetic and postmortem studies, all indicate that PSD proteins may be regarded as potential key molecules in the pathophysiology of complex disorders of synaptic plasticity and are worth further studies for elucidating their role in human behavior pathologies.

References

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