Glutamatergic Postsynaptic Density Protein ...

Mol Neurobiol DOI 10.1007/s12035-013-8534-3

Glutamatergic Postsynaptic Density Protein Dysfunctions in Synaptic Plasticity and Dendritic Spines Morphology: Relevance to Schizophrenia and Other Behavioral Disorders Pathophysiology, and Implications for Novel Therapeutic Approaches Andrea de Bartolomeis & Gianmarco Latte & Carmine Tomasetti & Felice Iasevoli

Received: 6 July 2013 / Accepted: 13 August 2013 # Springer Science+Business Media New York 2013

Abstract Emerging researches point to a relevant role of postsynaptic density (PSD) proteins, such as PSD-95, Homer, Shank, and DISC-1, in the pathophysiology of schizophrenia and autism spectrum disorders. The PSD is a thickness, detectable at electronic microscopy, localized at the postsynaptic membrane of glutamatergic synapses, and made by scaffolding proteins, receptors, and effector proteins; it is considered a structural and functional crossroad where multiple neurotransmitter systems converge, including the dopaminergic, serotonergic, and glutamatergic ones, which are all implicated in the pathophysiology of psychosis. Decreased PSD-95 protein levels have been reported in postmortem brains of schizophrenia patients. Variants of Homer1, a key PSD protein for glutamate signaling, have been associated with schizophrenia symptoms severity and therapeutic response. Mutations in Shank gene have been recognized in autism spectrum disorder patients, as well as reported to be associated to behaviors reminiscent of schizophrenia symptoms when expressed in genetically engineered mice. Here, we provide a critical appraisal of PSD proteins role in the pathophysiology of schizophrenia and autism spectrum disorders. Then, we discuss how antipsychotics may affect PSD proteins in brain regions relevant to psychosis pathophysiology, possibly by controlling synaptic plasticity and dendritic spine rearrangements through the modulation of glutamateA. de Bartolomeis (*) : G. Latte : C. Tomasetti : F. Iasevoli Laboratory of Molecular and Translational Psychiatry, Unit of Treatment Resistant Psychosis, Department of Neuroscience, Reproductive and Odontostomatologic Sciences, Section of Psychiatry, University School of Medicine “Federico II”, Via Pansini 5, 80131 Naples, Italy e-mail: [email protected]

related targets. We finally provide a framework that may explain how PSD proteins might be useful candidates to develop new therapeutic approaches for schizophrenia and related disorders in which there is a need for new biological treatments, especially against some symptom domains, such as negative symptoms, that are poorly affected by current antipsychotics. Keywords Psychosis . Antipsychotics . Synapse . PSD-95 . Homer . Shank

Introduction Schizophrenia is a complex disorder affecting nearly 1 % of the general population. A large amount of evidence suggests that schizophrenia is caused by aberrant synaptic plasticity and metaplasticity. Perturbation of regular dendritic spines architecture and function has been described in the disease [1–3]. Multiple neurotransmitter systems have been implicated in schizophrenia pathophysiology, and strong evidence points out abnormalities in dopamine, glutamate, and serotonin neurotransmission [4, 5]. Signaling pathways activated by these neurotransmitters converge on the postsynaptic density (PSD), which is considered as a structural and functional multi-protein crossroad. PSD is an electron-dense thickening localized under postsynaptic membranes, which comprises several hundred proteins and particularly characterizes large excitatory glutamatergic synapses [6]. PSD proteins are distributed in highly organized macromolecular complexes that process, integrate, and converge synaptic signals to the nucleus [7]. Overall, PSD proteins are involved in synaptic

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plasticity and dendritic spines architecture. Rearrangements in PSD proteins multimers at synaptic spines, occurring with precise stimulus-related spatiotemporal patterns, are currently supposed to underlie synaptic plasticity-related events, such as long-term potentiation (LTP) and long-term depression (LTD) [8, 9]. According to the biological functions of PSD in humans, a recent study demonstrated that mutations in 199 human PSD genes (the 14 % of all PSD genes) are implicated in more than 200 diseases [10]. The 50 % of these diseases are primary nervous system disorders, including neurological, psychiatric, and developmental disorders [10]. Moreover, a large part of those mutations may principally affect cognitive and learning processes, as well as emotion/affective behaviors and social interaction. Therefore, according to this view, emerging evidence is accumulating that implicates PSD protein dysfunctions in major psychiatric disorders in which cognitive/ behavioral and social processes are impaired, such as schizophrenia and autism spectrum disorders. Based on the N -methyl-D -aspartate (NMDA) receptor hypofunction hypothesis of schizophrenia [11], several studies have found abnormalities in PSD proteins in brain regions where NMDA receptors are localized, as well as gene association studies have revealed an increased risk for schizophrenia in patients with mutations in genes affecting NMDA functions (for a review, see [12]). Moreover, several molecular defects in PSD components and in PSD-related proteins have been found in postmortem brains of schizophrenia patients, mostly in regions implicated in the pathophysiology of the disease [13, 14]. Thus, PSD alterations may contribute to the synaptic derangements in schizophrenia [9]. The recent evidence that master organizing PSD scaffolding proteins are linked to autism spectrum disorders (ASD) [15] further confirms their role in synaptic plasticity processes potentially involved in cognition and social interaction. Consistent with the putative role of PSD proteins in schizophrenia and autism spectrum disorders, several studies have implicated glutamatergic PSD components in the molecular mechanisms of action of antipsychotic drugs. Though primarily acting on dopamine transmission, evidence exists that antipsychotics may also modulate glutamate-related targets, in particular the N-methyl-D -aspartate receptor (NMDAR)interacting molecules of the PSD [16, 17]. Indeed, different antipsychotic drugs have been shown to modulate glutamaterelated molecules in PSD [16, 18–22]. Therefore, PSD proteins have been proposed as a target for antipsychotic action [23]. Antipsychotics may affect glutamatergic neurotransmission at multiple levels, and they may regulate dendritic spine formation and synaptogenesis [24, 25], as well as the expression, trafficking, and functioning of PSD-related molecules [22, 26–28]. The aim of this review is to provide a critical description of PSD proteins role in the pathophysiology and treatment of

schizophrenia and other behavioral disorders, such as ASD. Herein, we will discuss how antipsychotics may affect PSD proteins in brain regions relevant to psychosis pathophysiology, possibly by controlling dendritic spines formation and synaptic plasticity through the modulation of specific NMDAR-related targets. Moreover, we will provide recent information on how PSD molecules may be considered valuable candidates to develop potential new therapeutic approaches for schizophrenia and autism spectrum disorders.

Clinical and Preclinical Evidence of PSD Proteins Involvement in Schizophrenia and other Behavioral Diseases PSD-95 PSD-95 Regulates Activity-Dependent Synaptic Shape and Strength PSD-95/SAP90 is a member of the membrane-associated guanylate kinase family (MAGUK), which has master organizing roles in the multimerization and clustering of protein complexes within the PSD (Fig. 1). PSD-95 protein contains in its structure three repeated PDZ (PSD-95/disc large/zonula occludens-1) domains, one SH3 (Src homology 3), and one guanylate kinase (GUK) domain [5]. PDZ domains are peptide-binding domains located at the C-terminus of MAGUK proteins, which may enable PSD-95 to interact with several binding partners within the PSD, such as NMDAR and 5HT receptor subunits, as well as other tyrosine kinase receptors and ion channels, cell adhesion molecules, and cytoplasmic proteins [29]. SH3 and GUK are other peptide-binding domains, which may also form intramolecular bonds [5]. Thus, by assembling in multimers with other PSD proteins, PSD-95 enables the formation of extensive protein complexes that organize receptors and signal transduction proteins in the PSD. Indeed, affinity-purified PSD-95 complexes have been described to include 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid receptor (AMPAR) subunits (GluR1, GluR2, GluR3, GluR4), NMDAR subunits (NR1, NR2A, NR2B), scaffolding proteins (PSD-93, Shank2, Shank3, Homer, SAPAP1, SAPAP2, SAPAP4), G protein regulators (such as SynGAP or BRAG1), and other PSD proteins [30, 31]. Furthermore, PSD-95 may interact with dopamine D2 and serotonin 5-HT2 receptors to regulate their activation state [5]. PSD-95 has also been described to stabilize glutamate receptors in the PSD and to provide a link between NMDARs and intracellular signaling molecules [9]. Given its functions, PSD-95 has been implicated in synaptic plasticity processes and in the interplay among glutamatergic, dopaminergic, and serotonergic signaling pathways. Hence, aberrant PSD-95 functioning may cause abnormal glutamate

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Fig. 1 Complex interactions among transductional pathways in the PSD. PSD proteins elaborate and integrate multiple transductional pathways starting at different membrane receptors (i.e., glutamate, dopamine). Scaffolding proteins (Homer, Shank, PSD-95) provide physical connections among different receptors, such as ionotropic and metabotropic glutamate receptors, as well as they link these receptors to intracellular calcium stores. Dopamine receptors activate transductional pathways that tightly intermingle with glutamatergic ones, through the action of key PSD proteins, such as GSK3, which may participate in the elaboration of diverse signals (dopamine, glutamate, Wnt) and regulate neuronal survival and differentiation. All these transductional pathways converge in the end on appropriate nuclear targets via specific effectors, such as CaMK, MAPKs, or Erk, in order to fine modulate long-term activity dependent neuronal rearrangements. NMDAR, N -methyl-D -aspartate

glutamate receptor; AMPAR , α-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid glutamate receptor; mGluR1a/5, metabotropic glutamate receptor type 1a/5; TARP, transmembrane AMPA receptors regulating protein or stargazin; PSD-95, postsynaptic density protein 95 kD; DISC1, disrupted in schizophrenia 1; GSK3, glycogen synthase kinase 3; PDE4, phosphodiesterase 4; GKAP, guanylate kinase-associated protein; H1a, Homer1a immediate-early inducible protein; PIP2, phosphatydilinositol bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; cAMP, cyclic adenosine monophosphate; ER, endoplasmic reticulum; PLC, phospholipase C; PKC, protein kinase C; PKA, protein kinase A; CAMK, calcium-calmodulin-regulated kinase; MAPKs, mitogen-activated protein kinases; Erk, extracellular signal-regulated kinase; MEK, MAPK/Erk kinase; Rac1, Ras-related C3 botulinum toxin substrate 1

signaling, thereby potentially taking part in molecular dysfunctions involved in schizophrenia and behavioral disorder pathophysiology. Fine-tuned PSD proteins interactions by PSD-95 may contribute to long-term changes in synaptic shape and strength. In CA1 hippocampal pyramidal neurons, activity-dependent growth of apical spines has been associated with the destabilization of PSD architecture, causing transient loss and rapid replacement of PSD-95, together with Shank2 [32]. It has also been observed that signaling through a PSD-95-mediated pathway is required for activity-dependent synaptic enlargement and for the recruitment of other architectural proteins, such as Shank [32]. PSD-95 has been supposed to participate in multiple steps of synaptic rearrangements, based on the discovery that

different PSD-95 domains have been found implicated in this process [32]. Morphological changes in PSD by PSD95 may be regulated through CaMKII phosphorylation at Ser73 site, which has been reported to slow down both the growth of apical spines and the strength of synaptic currents [32]. Therefore, PSD-95 may both trigger synaptic growth and terminate it, probably via the modulation of growthrelated proteins trafficking in the PSD. Accordingly, the knockdown of PSD-95 gene expression by short hairpin RNA has been shown to impair early and late phases of spine growth [32]. The overexpression of PSD-95 may affect spines morphologybyincreasingspinevolumeandexpandingPSDstructure,as well as by concomitantly decreasing spines density [33]. PSD95 overexpression may also induce the formation of multi-

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innervated spines, i.e. dendritic spines that are connected with up to seven presynaptic terminals [33]. The formation of multiinnervated spines is prevented by deletion of the nitric oxide synthase(NOS)-interactingPDZ2 domainofPSD-95[33].Ithas been reported that NOS–PSD-95 interaction and nitric oxide (NO) signaling may promote synapse formation. Downregulation by small interfering RNA or by pharmacological blockade of NOS may prevent the formation of multiple synapses mediated by PSD-95 overexpression [33]. Nevertheless, treatment of hippocampal slices either with a NO donor or with cyclic guanosine monophosphate analogues has been demonstrated to induce the formation of multi-innervated spines by PSD-95 overexpression [33]. Recent studies have demonstrated that PSD-95, in cooperation with other PSD scaffolding proteins (i.e. Shank, Homer1, GKAP), may establish the architectural basis for glutamate receptor clustering—in particular of AMPARs—in response to prolonged stimuli [34]. In particular, PSD-95 might have a crucial role in concentrating AMPARs in postsynaptic active zones in correspondence of presynaptic glutamate-releasing sites, thereby maximizing the effects of postsynaptic excitatory potentials [31]. Therefore, PSD-95 enrichment within PSD may regulate morphological and functional synaptic plasticity in multiple and complex ways. Actually, PSD-95 may trigger synaptic growth, and its overexpression may block the growth of single synapses and favor the formation of multi-innervated spines. PSD-95 interactions may drive postsynaptic architectural remodeling in response to stimuli. Moreover, together with the other scaffolding proteins, PSD-95 may modify glutamate receptors postsynaptic membrane position, thus increasing or decreasing excitatory currents even without altering the number of receptors. PSD-95 Bridges Dopamine, Glutamate, and Serotonin Transductional Pathways PSD-95-related proteins provide a physical link between glutamate and dopamine systems. PSD-95 proteins may regulate dopamine D1 receptor (D1R) trafficking and function, through the interaction between the carboxyl-terminal tail of D1Rs and the NH(2) terminus of PSD-95 [35]. PSD-95 may reduce surface D1R expression by promoting receptor internalization [35, 36]. Co-expression of PSD-95 and D1Rs in mammalian cells has been found to inhibit D1R-mediated cAMP accumulation [35]. Therefore, the overexpression of PSD-95 may reduce D1R signaling and prevent functional hyperdopaminergia. Accordingly, genetically engineered mice lacking functional PSD-95 proteins exhibit increased response to direct D1R agonists or to indirect dopamine agonists (e.g. amphetamine) [35]. PSD-95 may be also implicated in preventing the concurrent overactivation of D1Rs and NMDARs during neurotoxicity states. Indeed, PSD-95

proteins have been demonstrated to inhibit D1R-NMDAR association and uncouple the NMDAR-dependent enhancement of D1R signaling [36]. Thus, PSD-95 gene expression may inhibit D1R function, whereas its knockdown may enhance NMDAR-dependent D 1 R functioning [35, 36]. Notably, the disruption of the interaction between D1Rs and PSD-95 in striatum has been described to reduce L -DOPAinduced dyskinesia in rat and macaque models [37], thereby suggesting the involvement of PSD-95/D1R complexes in dopamine-related dysfunctions. Besides dopamine D1Rs, PSD-95 has been demonstrated also to physically interact with dopamine D2Rs in cell cultures [38]. However, the in vivo effects of PSD-95/D2R interaction are still elusive at present. In the striatum, D1Rs and D2Rs are expressed in functionally different neuronal populations, being D1Rs selectively expressed in the medium-sized spiny neurons of the direct pathway and D2Rs in the indirect pathway cells [39]. Provided the inhibitory effects of PSD-95 binding to D1Rs on the NMDAR/D1R downstream signaling, it is possible that PSD-95 might exert “D2-like” slowing down functions on D1R signaling in striatal neuron subtypes in which D2Rs are not physically expressed. PSD-95 interacts with Calcyon, a transmembrane protein predominantly expressed in the central nervous system [40] that may be increased in schizophrenia patients [41, 42]. PSD95 and Calcyon have been found to form a ternary complex with D1Rs in dendritic spines of hippocampal neurons [43]. Furthermore, the PKC-dependent phosphorylation of Calcyon may promote its association with PSD-95 and the recruitment to plasma membrane, as well as it may enhance the internalization of surface D1Rs [43]. Thus, the Calcyon–PSD–95D1R complex may represent a further mechanism linking dopamine and glutamate transductional pathways and also a potential target for psychopharmacotherapy. PSD-95 is also essential for proper targeting and synaptic membrane stabilization of serotonin 5-HT2A and 5-HT2C receptors. The interaction with PSD-95 may prevent agonistmediated internalization of 5-HT2A receptors and enhances 5HT2A receptor-dependent signaling [44], as well as it may promote 5-HT2C constitutive and agonist-mediated internalization [45]. PSD-95 has been reported to regulate 5-HT2A receptors surface membrane turnover, thus contributing to their targeting to apical dendrites of pyramidal neurons. PSD-95 is also required for 5-HT2C-mediated signaling in vivo, as well as for 5-HT2A-mediated actions of hallucinogenic drugs [46]. Notably, both 5-HT2A and 5-HT2C receptors downstream pathways, as well as the animal behaviors mediated by the activation of these receptors, have been found impaired in mice lacking functional PSD-95 [46]. Also, treatment with either clozapine (the prototypical “atypical” antipsychotic compound), M100907 (a 5-HT2A receptor antagonist), or SR46349B (a 5-HT2A/2C antagonist), all of which may regularly reduce behavioral impairments induced by

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NMDAR-blocking drugs (such as phencyclidine), is ineffective in PSD-95 null mice [46]. By controlling 5-HT2A/2C receptor trafficking and signaling, PSD-95 may contribute to the mechanisms of action of both hallucinogenic drugs and atypical antipsychotics. Hence, PSD-95 may act as a pivotal crossroad molecule along dopamine, glutamate, and serotonin signaling systems and enable their interplay and reciprocal transactivation. PSD-95 in the Pathophysiology of Psychotic Disorders A growing number of studies have implicated PSD-95 in animal models of psychosis, even if with the limitation of animal modeling in psychiatry, and in the pathophysiology of schizophrenia, as demonstrated by postmortem human brain analyses (Table 1). In animal studies, perinatal phencyclidine (PCP) administration in rats has been demonstrated to lead to complex changes in PSD-95 protein expression in both prefrontal cortex (PFC) and hippocampus throughout the central nervous system development. The injection of PCP in rats at postnatal days 7, 9, and 11 may provoke alterations in NMDAR subunits, in neuregulin1/erbB4 expression levels, and in erbB4 phosphorylation that may be detected immediately after the injection (postnatal day 12) and last until 20 weeks after birth, thus providing new insights on abnormal developmental processes putatively involved in schizophrenia [47]. Nevertheless, neuregulin1 may increase PSD-95 protein levels and stability; these effects being specific to GABAergic interneurons [47]. Decreased PSD-95 protein expression has also been found in the PFC of isolation-reared female rats, an animal model of neuropsychiatric disorders that include features reminiscent of anxiety- and schizophrenia-like disorders [48]. In human postmortem studies, altered PSD-95 gene and protein expression have been reported in brain regions involved in schizophrenia pathophysiology. Early works demonstrated an increase in thalamic PSD-95 gene expression in schizophrenia patients, with a concurrent decrease in NMDAR NR1 subunits in the same region [14]. Noteworthy, more recent studies by the same group have described opposite regulation of PSD-95 transcripts in the thalamus of schizophrenia patients, depending on the disease onset age. Postmortem studies indicated that young schizophrenia patients have decreased PSD-95 thalamic levels and increased NMDAR NR2B subunits [13], whereas in elderly patients, increased PSD-95 and NR2B protein levels in dorsomedial thalamus were found [49]. In the anterior cingulate cortex, increased expression of PSD-95 transcripts but decreased protein expression and decreased phosphorylation at Ser295 site have been reported [29, 50, 51]. Decreased PSD-95 protein expression has also been described in the dentate molecular layer of hippocampus [52] and in a subcellular endoplasmic reticulum-enriched fraction from the

dorsolateral prefrontal cortex [53] of schizophrenia patients. Thus, both animal and human studies provide substantial evidence of a crucial role of PSD-95 in the normal establishing of correct glutamatergic synaptic downstream signaling during neurodevelopment. Early alterations in PSD-95 functions may lead to aberrant glutamatergic signaling that is putatively at the basis of cognitive and behavioral dysfunctions in psychotic disorders. Based on the putative role of PSD-95 in spine enlargement and synaptic strength, it could be hypothesized that lack of functional PSD-95 may cause dendritic spine shrinking and abnormal synaptic plasticity, which may contribute to the molecular underpinnings of psychosis. According to this view, it has been observed that pluripotent stem cells from peripheral fibroblasts of schizophrenia patients may show decreased PSD-95 protein amounts and reduced neuronal connectivity when differentiated into neurons [19]. On the other hand, enhanced PSD-95-ErbB4 coupling has been found in PFC of schizophrenia patients [54], thereby suggesting that enhanced interactions between these two molecules may be involved in schizophrenia pathophysiology. ErbB4 is a member of the epidermal growth factor receptors family of tyrosine kinase receptors, which are essential for normal nervous system development [55, 56]. Despite the biological role of PSD–95-ErbB4 interaction has not been yet elucidated, an increased coupling of these two molecules may be responsible for ErbB4 signaling blockade (i.e. by promoting ErbB4 recycling and endocytosis) or be secondary to synaptic derangements in schizophrenia. However, discrepant findings in postmortem studies may derive from several technical and methodological limitations, including the potential effects of chronic antipsychotic treatment, differences in the type of prevailing symptoms (positive, negative, or cognitive ones), total duration of the illness, patients’ kind of death, and elapsed time between death and storage of the specimen [57, 58]. Consistently with its putative role in cognitive and behavioral disorders pathophysiology, PSD-95 has also been implicated in other neurodevelopmental diseases, such as autism. Mice with a homozygous PSD-95 gene deletion (Dlg4 −/− ) show a complex phenotype reminiscent of autism, which includes increased repetitive behaviors, abnormal communication and social behaviors, impaired motor coordination, increased stress- and anxiety-related responses [59]. These mutant mice also exhibit altered dendritic spine morphology in amygdala and altered forebrain gene expression profile [59]. Moreover, prenatal rat exposure to valproic acid, a model of ASD, may lead to increased PSD-95 protein expression in male but not female rats in hippocampus and cortex [60]. These results seem in agreement with multiple studies in which male preponderance in risk factors for ASD is suggested. However, neither the hypothesis of excessive fetal testosterone, nor of Y chromosome-linked abnormalities, nor

Mol Neurobiol Table 1 Clinical and preclinical evidence of PSD-95 modulation by dopaminergic and glutamatergic stimuli and of its implication in the pathophysiology of psychotic disorders Experimental paradigm/ sample

Drug

Dose and Technique/assessment route of administration

PSD-95 overexpression in hippocampal slice cultures

L-NG-nitroarginine methyl ester 200 μM (L-NAME, nitric oxide in vitro; synthase inhibitor); 150 μM diethylene-triamine NONOate in vitro; (NO donor); 8-Br-cGMP 5 mM (cGMP analogue) in vitro

Immunohistochemistry, electron microscopy

Post-weaning social – isolation in female rats

–

Adm. on postnatal days 7, 9, and 11 in rats

10 mg/kg s.c. Western blot (PSD-95 protein)

PCP

Western blot, behavioral tests (open field test, novelty suppressed feeding, social interaction test)

Outcome

Reference

PSD-95 overexpression affects spine morphology and promotes multi-innervated spine formation: formation of multiple contacts is prevented by deletion of the nitric oxide– interacting domain PDZ2 PSD-95 overexpression associated with nitric oxide synthase down-regulation or blockade prevents axon differentiation: treatment with nitric oxide donor or cyclic guanosine monophosphate analogue induces multiinnervated spine formation Decreased PSD-95 (and synapsin I, GluR1, NR1) expression in the prefrontal cortex. Deficits in social behaviors and increased anxiety-like behavior Increased expression at 5 weeks (with return to control level at 20 weeks) in prefrontal cortex increased expression at postnatal day 12 and at 5 weeks, and decreased expression at 20 weeks in hippocampus associated changes in NMDAR subunits, neuregulin1/erbB4 expression levels, erbB4 phosphorylation NRG1 increases the level and stability of PSD-95 protein in GABAergic interneurons in a manner that requires tyrosine kinase activity of ErbB4 No changes in caudate-putamen and n. accumbens No changes in cortex, caudateputamen, and n. accumbens Decreased expression in MAC, MC, n. accumbens core

[33]

NRG1 (5 nM) applied to – dissociated cortical neurons

–

Acute administration. in rat

Haloperidol Olanzapine Quetiapine

1 mg/kg i.p. ISHH (PSD-95 mRNA) 0.5 mg/kg i.p. ISHH (PSD-95 mRNA) 30 mg/kg i.p. ISHH (PSD-95 mRNA)

Haloperidol

0.8 mg/kg i.p. ISHH (PSD-95 mRNA)

Haloperidol + valproate

0.8 + 500 mg/ ISHH (PSD-95 mRNA) Increased expression in ACC, kg i.p. SS, dmCP 30 + 500 mg/ ISHH (PSD-95 mRNA) No changes in cortex, caudatekg i.p. putamen and n. accumbens 15 mg/kg i.p. ISHH (PSD-95 mRNA) decreased expression in MAC, MC 14 mg/kg i.p. ISHH (PSD-95 mRNA) Increased expression in ACC, MAC, SS, IC, all subregions of caudate-putamen, n. accumbens core, and shell 30 mg/kg i.p. ISHH (PSD-95 mRNA) No changes in cortex, caudate0.8 mg/kg i.p. ISHH (PSD-95 mRNA) putamen, and n. accumbens ISHH (PSD-95 mRNA)

Quetiapine + valproate Quetiapine Citalopram

16 days administration in Quetiapine Haloperidol rat Haloperidol + valproate

Immunocytochemistry, western blot, electrophysiological recordings

[48]

[47]

[177]

[150] [178]

Mol Neurobiol Table 1 (continued) Experimental paradigm/ sample

Drug

Quetiapine + valproate

21 days administration in rat

Quetiapine Citalopram Haloperidol

Ziprasidone

Sertindole Acute administration in PSD-95 null mice

Phencyclidine + clozapine

miR-485 overexpression in hippocampal neurons in culture

–

miR-125a transfection, antimiR-125a transfection. Hippocampal neurons in culture, Neuro2A, and L-M(TK-) cells

Dihydroxyphenylglycine (DHPG, group I mGluRs agonist)

Human postmortem – brains of schizophrenia patients Human postmortem – brains of young schizophrenia patients Human postmortem – brains of elderly schizophrenia patients Human postmortem – brains of schizophrenia patients

Human postmortem – brains of schizophrenia patients

Dose and Technique/assessment route of administration 0.8 + 500 mg/ kg i.p. 30 + 500 mg/ kg i.p. 15 mg/kg i.p. 14 mg/kg i.p. 0.8 mg/kg i.p.

Outcome

Reference

ISHH (PSD-95 mRNA)

ISHH (PSD-95 mRNA) ISHH (PSD-95 mRNA) ISHH (PSD-95 mRNA) Increased expression in the cortex (MAC, MC, SS), all subregions of caudateputamen, n. accumbens core and shell 4 mg/kg i.p. ISHH (PSD-95 mRNA) Increased expression in ACC, all subregions of caudateputamen, n. accumbens core and shell 2 mg/kg s.c. ISHH (PSD-95 mRNA) No changes in cortex, caudateputamen, and n. accumbens 0.5 + 6.0 mg/kg PPI assessment Genetic deletion of PSD-95 abolishes clozapine-mediated normalization of PCP-induced PPI deficit – Immunocytochemistry miR-485 overexpression reduces (PSD-95 protein) spine density, increases immature spines, and reduces PSD-95 clustering 50 μM Fluorescence in situ PSD-95 translation is in vitro hybridization, bidirectionally regulated immunocytochemisthrough the control of both try, miR-125a and the fragile X immunoprecipitation, mental retardation protein western blot, (FMRP) phosphorylation luciferase assay status – ISHH (PSD-95 mRNA) Increase in thalamic PSD-95 expression, with a concurrent decrease in NR1 subunit of NMDA receptors – ISHH (PSD-95 mRNA) Decrease in thalamic PSD-95 expression, with a concurrent increase in NR2 subunit of NMDA receptors – Western blot analysis Increase in thalamic protein levels of PSD-95 and of NR2 subunit of NMDA receptors – Western blot analysis Decreased protein levels of PSD95 and NR2 subunit of NMDA receptors in dorsolateral prefrontal cortex and anterior cingulate cortex – Immunoautoradiography Decrease in PSD-95 protein levels in the dentate molecular layer of hippocampus, not related to antipsychotic treatment

[16]

[22] [46]

[120]

[151]

[14]

[13]

[49]

[51, 53]

[52]

ACC anterior cingulate cortex; cGMP cyclic guanosine monophosphate; dlCP, vlCP, dmCP, vmCP dorsolateral, ventrolateral, dorsomedial, and ventromedial caudate-putamen respectively; IC insular cortex; ISHH in situ hybridization histochemistry; MAC medial agranular cortex; MC motor cortex; NO nitric oxide; PPI prepulse inhibition; RT-PCR reverse transcription polymerase chain reaction; SS somatosensory cortex

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of male vs. female differences in social empathizing functions may fully explain the prominent male ASD prevalence (for a review, see [61]). Recent results confirm the higher threshold of female susceptibility to ASD as compared to males. Sato and colleagues [62] recently reported that Shank1 mutations associated with ASD phenotypes are limited to males. These studies reinforce the view that alterations of PSD-95 may cause defective synaptic architecture and spine functions. Interestingly, different genes linked to autism (such as protocadherin 10, Pcdh10; fragile X mental retardation 1, Fmr1; myocyte enhancer factor 2, Mef2) may participate in the degradation of PSD-95 and in synapse elimination, thus suggesting a pivotal function of PSD-95-mediated deficits in synapse elimination among the different genetic causes of autism [63]. PSD-95 dysfunctions have also been implicated in Angelman syndrome (AS), a neurodevelopmental disorder that includes cognitive impairment and autism. Ubiquitin-protein ligase E3A knockout mice (a model of AS) exhibit high Arc protein amount in response to synaptic activity, impaired LTP in the hippocampus, and deficits in learning behaviors [64]. Impaired LTP may be due to a reduction in BDNF-induced PSD-95 association with TrkB, thereby resulting in reduced PLCγ and PI3K signaling [64]. The administration of CN2097, a PSD-95 binding peptide interacting with the PDZ domain, may reduce Arc/PSD-95 interactions, thus restoring BDNF-induced TrkB/PSD-95 complex formation and LTP induction [64]. Taken together, these findings suggest that PSD-95 may regulate synaptic targeting and localization of several PSD proteins, which are organized as a “molecular lego” in the architecture of dendritic spines. Moreover, PSD-95 may represent an intermediate molecule along multiple neurotransmitter systems, allowing their cross-talk at both structural and functional levels. These observations provide support for studying PSD-95 as a candidate molecule to better dissect the pathophysiological bases of cognitive impairment in neuropsychiatric disorders, such as schizophrenia or ASD. Homer Homer Proteins Are Multimodal Postsynaptic Adaptors that Fine Regulate PSD Architecture Homer genes encode for a family of proteins including three isoforms in mammals (Homer1, Homer2, and Homer3), which are predominantly localized at the PSD, where they act as multimodal adaptors by interacting with several PSD proteins [7, 65, 66] (Fig. 1). Homer proteins are primarily classified into (a) constitutively expressed isoforms (i.e. Homer1b/c, Homer 2, and Homer 3), which are bimodal proteins with a N-terminal Ena/VASP (EVH) domain allowing the binding to other PSD proteins, and a C-terminal

coiled-coil domain that enables self-assembly; (b) short, nonmultimerizing, activity-dependent splice variants of the Homer1 gene (Homer1a, Ania-3), which lack the C-terminal domain and are able to interact with PSD targets but cannot self-assemble [65, 66]. These short forms are induced in an immediate-early gene-like fashion after neuronal stimulation and act as endogenous “dominant-negative” by disrupting long Homer isoforms protein–protein interactions [65]. Once induced, Homer short forms cause rapid and transient rearrangements of long Homer clusters and in turn of synaptic architecture. These transient changes have significant consequences for synaptic signaling, including agonist-independent activation of group I metabotropic glutamate receptors (mGluRs) [67], changes in local Ca2+ levels [68], activation/ inactivation of surface ion channels [69], and modulation of second messenger signaling [9, 70]. The relative ratio of long/short Homer forms (i.e. Homer1b/c vs. Homer1a) has also been described to impact ion channel functioning and synaptic signaling [66], as well as synaptic architecture, spine shape, and size. Indeed, Homer1b may enhance Shank1B-mediated increase in spine length and width [71]. On the other hand, overexpression of Homer1a may reduce the number and size of dendritic spines, decrease the density of postsynaptic proteins (such as Shank) in spines, and inhibit postsynaptic AMPAR and NMDAR currents in hippocampal neurons [72]. Homer1a is instrumental in the activity-induced reorganization of both pre- and postsynaptic structures. Basically, Homer1a induction is involved in glutamate-induced biphasic changes in the distribution of both presynaptic proteins (such as synaptotagmin, synaptophysin, and synapsin) and postsynaptic proteins (such as PSD-95 and Homer1c), at least in cultured hippocampal neurons [73]. Furthermore, Homer1a protein induction may reduce group I mGluR-dependent LTD in layer VI pyramidal cells from rat visual cortex slices [74]. Finally, the acute disruption of mGluR–Homer interactions has been demonstrated to impair mGluR-dependent LTD in rat hippocampal slices [75]. Long and short Homer isoforms may cooperate to fine tune PSD-mediated synaptic plasticity. This fine tuning appears stringently regulated in space (i.e. in PSD microdomains) and time (i.e. short Homer proteins are induced to provide time-limited rearrangements of long Homer clusters). Homer Proteins Modulation by Dopaminergic and Glutamatergic Stimuli Several studies have demonstrated that the expression of transcript encoding for both long and short Homer isoforms may be affected by psychotomimetic drugs modulating either dopaminergic or glutamatergic receptors. Both acute methamphetamine and cocaine administration may induce Homer1a mRNA in the neocortex of saline-pretreated rats [76]. Also,

Mol Neurobiol

increased long Homer protein amount in the nucleus accumbens has been described after acute cocaine administration [77]. Consistent with the role of Homers as scaffold proteins at the crossroad of dopamine and glutamate system, drugs acting at NMDARs, such as the noncompetitive inhibitor PCP, may increase Homer1a mRNA expression in rat PFC prelimbic region and in primary auditory cortex 2 h after treatment, as well as it may decrease Homer1a mRNA expression in retrosplenial cortex and dentate gyrus 24 h after treatment [78]. Similarly to PCP, the NMDAR noncompetitive antagonist ketamine may induce Homer1a mRNA in the ventral striatum and in the core and the shell of the nucleus accumbens when acutely administered at subanesthetic doses [79]. Both PCP and ketamine are NMDAR-noncompetitive antagonists known to cause psychotic symptoms in humans and to exacerbate psychotic symptoms in schizophrenia patients [80, 81]. Moreover, PCP and ketamine are believed to mimic NMDAR hypofunction, which is considered a valuable and heuristic pharmacological model of schizophrenic symptoms [14]. Homer in the Pathophysiology of Psychotic Disorders Given its role in activity-dependent synaptic rearrangements, Homer gene and protein expression changes in response to dopaminergic and glutamatergic stimuli have been considered as fine-tuned mechanisms to preserve synaptic homeostasis [82]. Thus, growing evidence has been provided that Homer protein dysfunctions might be involved in the pathophysiology of neuropsychiatric disorders implicating defects in synaptic plasticity, such as schizophrenia [83, 84] (Table 2). The deletion of Homer1 gene has been demonstrated to induce behavioral and neurochemical abnormalities relevant to animal models of schizophrenia, including altered performances in sensory, motor, social, and learning/memory tests [85, 86]. Homer1 knockout (KO) mice show altered food reward and reinforcement, altered antipsychotic-sensible sensorimotor gating, increased motor activation, and attenuated habituation of motor activity when exposed to a novel environment. These mice also have increased sensitivity to the locomotoractivating effects of MK-801 or methamphetamine, decreased extracellular glutamate content in the nucleus accumbens, and increased content in the PFC, as well as blunted increase in PFC extracellular glutamate after cocaine stimulation [86]. However, long and short Homer1 variants are differently involved in PFC glutamate neurotransmission and in development of behaviors relevant to schizophrenia. Actually, a recent study demonstrated that the adeno-associated virus (AAV)-mediated restoration of either Homer1a or Homer1c in Homer1 KO mice may differently affect synaptic functions and consequent behaviors [87]. Homer1c restoration in the PFC of Homer1 KO mouse reverses aberrant working memory and sensorimotor function, locomotor hyperactivity in

response to a novel environment, sensitivity to cocaine, and PFC glutamate content [87]. By contrast, the AAV-mediated restoration of Homer1a has been demonstrated to only reverse alterations in emotional reactivity in mutant animals [87]. Notably, these behavioral and neurochemical alterations have been found worsened by dopamine function enhancers, such as cocaine and methamphetamine, and were prevented or attenuated by D2 receptor antagonist agents, such as haloperidol. Consistent with preclinical findings, several clinical studies have implicated Homer genes in schizophrenia pathophysiology. An early study found a significant association between a single nucleotide polymorphism (SNP) within intron 4 of Homer1 gene and schizophrenia [88]. However, this result was not replicated in an extended sample by the same authors [88]. More recently, an association between Homer1 gene polymorphisms and clinical psychopathology assessments in schizophrenia has been demonstrated. Two Homer1 polymorphisms (rs2290639, which is an intronic polymorphism, and rs4704560, which is a mutation in the 5′-flanking region of Homer1 gene, and could be considered as a potential promoter polymorphism) have been associated with scores on positive and negative syndrome scale (PANSS, a rating scale for symptoms severity assessment in schizophrenia) subscales at baseline. Namely, the rs2290639 variant was significantly associated with scores on PANSS total, positive, and global psychopathology subscales, whereas the rs4704560 variant was significantly associated with scores on PANSS-negative subscale [89]. Also, a putative role for Homer2 gene in schizophrenia susceptibility has been suggested. Actually, the rs2306428 polymorphic variant has been strongly associated with the disease [90]. The role of Homer proteins in synaptic plasticity has stimulated further studies on their involvement in neuropsychiatric diseases in which molecular processes underlying cognition are considered dysfunctional, such as ASD. Homer1 gene has been recognized as a novel autism-risk gene in a single nucleotide variant analysis of blood samples from 290 unrelated nonsyndromic autism cases and 300 ethnically matched controls [91]. Several rare and potentially damaging variants have been identified in the autism population that co-segregate with the disorder and affect functionally relevant protein regions or regulatory sequences [91]. Intriguingly, the interaction between long Homer isoforms and mGluR5 is strongly diminished in Fmr1 KO mice, a model of fragile X syndrome (an inherited cause of intellectual disability and autism) [92]. In this model, the genetic deletion of Homer1a may restore the long Homer–mGluR5 interaction and correct altered phenotypes, thus suggesting a potential Homer-related mechanism of mGluR5 dysfunction in this autism-related disease [93].

Mol Neurobiol Table 2 Clinical and preclinical evidence of Homer1 modulation by dopaminergic and glutamatergic stimuli and of its implication in the pathophysiology of psychotic disorders Experimental paradigm/ sample

Drug


Outcome

Reference

Homer1 KO mice

–

–

Behavioral assessment

[85]

–

–

Behavioral assessment, in vivo microdialysis

Homer1 KO mice: adeno- – associated virus (AAV)mediated restoration of Homer1a or Homer1c

–

Behavioral assessment, in vivo microdialysis

An extensive range of disrupted behaviors and altered performance in sensory, motor, social, and learning/memory tests (including altered social behavior, altered performance in the swim test, altered nest-building behavior, altered water maze learning performance) Behavioral and neurochemical abnormalities including altered food reward and reinforcement, altered sensorimotor gating that responded to haloperidol pretreatment, altered acquisition of a radial arm maze task, reduced exploratory behavior directed toward novel objects, increased motor activation and attenuated habituation of motor activity when exposed to a novel environment, increased sensitivity to the locomotor-activating effects of MK-801 or methamphetamine, decreased extracellular glutamate content in the nucleus accumbens and increased content in the prefrontal cortex, blunted cocaine-stimulated increase in extracellular glutamate in the prefrontal cortex Restoration of Homer1a or Homer1c differently affects working memory, sensorimotor function, emotional reactivity responses, sensitivity to cocaine and prefrontal cortex glutamate content Homer1a induction in neocortex Homer1a induction in neocortex

Acute administration after 5 days—pretreatment with saline in rat Acute administration in mice Acute administration in rat

Methamphetamine 1.6 mg/kg i.p. Quantitative RT-PCR Cocaine 30 mg/kg i.p. Quantitative RT-PCR Cocaine

20 mg/kg i.p. Immunoblotting

Cocaine

5, 10, or 20 mg/kg i.p.

Western blot (Homer1a protein)

ketamine

12 or 50 mg/ kg i.p.

ISHH (Homer1a mRNA)

PCP

2.58 mg/kg i.p.

ISHH (Homer1a mRNA)

Haloperidol

0.8 mg/kg i.p. ISHH (Homer1a mRNA)

Increased long Homer protein in n. accumbens Cocaine 10 and 20 mg/kg induced a rapid and transient increase in Homer1a protein expression in the dorsal and ventral striatum. The increase was blocked by a D1 (SCH23390), but not a D2 (eticlopride) receptor antagonist Ketamine 12 mg/kg increased Homer1a mRNA expression in vmCP and in n. accumbens core and shell ketamine 50 mg/kg increased Homer1a mRNA expression in vmCP, vlCP, n. accumbens core and shell Increased Homer1a mRNA expression in prefrontal cortex prelimbic region and primary auditory cortex 2 h posttreatment decreased Homer1a mRNA expression in retrosplenial cortex and dentate gyrus 24 h posttreatment Homer1a induction in all subregions of caudate-putamen, n. accumbens core, and shell

[86]

[87]

[76]

[77] [152]

[79]

[78]

[16, 18, 21, 22,


Drug


Outcome

0.8 mg/kg i.p. ISHH (Homer1b/c mRNA)

Decreased Homer1b/c expression in the 155, cortex (MC, SS, IC), vlCP, n. 178] accumbens shell Homer1a induction in lateral regions of caudate-putamen and in n. accumbens core Homer1a induction in the cortex (ACC, MAC, SS, IC) and in n. accumbens core and shell Aripiprazole 12 mg/kg: Homer1a induction in all subregions of caudateputamen aripiprazole 30 mg/kg: Homer1a induction in frontal and cingulate cortex and in n. accumbens shell Quetiapine 15 mg/kg: no changes in Homer1a expression quetiapine 30 mg/kg: Homer1a induction in the cortex (ACC, MAC, MC) Homer1a induction in lateral regions of caudate-putamen Ziprasidone 4 mg/kg: Homer1a induction in all caudate-putamen subregions ziprasidone 10 mg/kg: Homer1a induction in the cortex (ACC, MC, SS, IC) and in all caudateputamen and n. accumbens subregions decreased Homer1a expression in the cortex (SS, IC) Quetiapine 15 and 30 mg/kg: decreased [178] Homer1a expression in the cortex (MAC, MC, SS) Increased Homer1a expression in all CP [16, 18, 21, subregions and in nucleus accumbens 153, core Increased Homer1b/c expression in the 179] cortex (ACC, MAC, SS, IC), dlCP, vmCP, vlCP, n. accumbens core, and shell increased Homer1a expression in frontal cortex Decreased Homer1a expression frontal, parietal, and cingulate cortex; no changes in Homer1b/c expression

olanzapine


Clozapine

15 mg/kg i.p. ISHH (Homer1a mRNA)

Aripiprazole


ISHH (Homer1a mRNA)

Quetiapine


ISHH (Homer1a mRNA)

risperidone

3 mg/kg i.p.

ISHH (Homer1a mRNA)

ziprasidone

4 or 10 mg/kg ISHH (Homer1a mRNA) i.p.

sertindole

2 mg/kg s.c.

ISHH (Homer1a mRNA)


Quetiapine


ISHH (Homer1a mRNA)


Haloperidol


0.8 mg/kg s.c. ISHH (Homer1b/c mRNA)

Olanzapine

2 mg/kg i.p.

DNA microarray

Clozapine

15 mg/kg

ISHH (Homer1a, Homer1b/ c mRNA)

Aripiprazole

12 mg/kg


Quetiapine Ziprasidone

15 mg/kg i.p. ISHH (Homer1a mRNA) 4 mg/kg i.p. ISHH (Homer1a mRNA)

Sertindole

2 mg/kg s.c.


–

Genotyping, weekly psychopathology

267 schizophrenic patients – treated in monotherapy

Reference

Increased Homer1a expression in dlCP, vlCP, decreased expression in frontal and cingulate cortex no changes in Homer1b/c expression No changes in Homer1a expression Increased Homer1a expression in lateral regions of CP and in n. accumbens core No changes in Homer1a expression, increased Homer1b/c expression in the cortex (ACC, MAC, SS, IC) Two Homer1 polymorphisms, rs2290639 [89] and rs4704560, associated with scores


Drug

Dose and Technique/assessment route of administration assessment using Positive and Negative Syndrome Scale (PANSS)

with different antipsychotics

DNA pools of 368 schizophrenia patients and related controls

–

–

Genotyping

Outcome

Reference

on PANSS subscales at baseline 7 polymorphisms associated with clinical response after different 4-week antipsychotic treatments One SNP (Homer 1 IVS4+18A>G) [88] associated with schizophrenia, a finding confirmed by individual SNP genotyping (P =0.01)

ACC anterior cingulate cortex; dlCP, vlCP, dmCP, vmCP dorsolateral, ventrolateral, dorsomedial, and ventromedial caudate-putamen, respectively; IC insular cortex; ISHH in situ hybridization histochemistry; MAC medial agranular cortex; MC motor cortex; NO nitric oxide; PPI prepulse inhibition; RTPCR reverse transcription polymerase chain reaction; SS somatosensory cortex

In summary, Homer proteins are key proteins of the PSD, involved in postsynaptic glutamatergic signaling and in dopamine glutamate cross-talk. Long Homers act as scaffolding proteins, bridging glutamate receptors with their intracellular effectors. Short Homers disrupt these clusters in a space- and time-controlled fashion. This balance contributes to finely regulate multiple biological functions, such as Ca2+ dynamics in dendritic spine microdomains, whose disruption may concur to dysfunctions of synaptic plasticity and aberrant behavioral manifestations [94].

Shank Shank Proteins Form Functional Protein Platforms at Postsynaptic Sites The ProSAP/Shank (named from SH 3 and ankyrin domains) family of proteins is constituted by master organizing PSD scaffolding proteins that are implicated in propagating and modulating glutamate neurotransmission [95] (Fig. 1). Shank genes derive from the same orthologous genes, with slight differences in humans and animals, thus providing and optimal candidate for translational research on its involvement in neuropsychiatric disorders [96, 97]. Shank proteins, i.e. Shank1, Shank2, and Shank3, are coded by three different genes, each of them having multiple splice variants producing different Shank proteins [98]. Shanks may cross-link Homer and PSD-95 in the PSD and participate in NMDAR, mGluR, and AMPAR downstream signaling [99–101]. Moreover, Shanks promote spine formation, as well as maturation and enlargement of dendritic spines [101]. Through their multiple interactions, Shank proteins enable the formation of a polymeric network complex, which requires assembly of Homer tetramers and Shank multimers. This structure has been proposed to serve as a functional platform for other PSD proteins [95].

Shank in the Pathophysiology of Psychotic Disorders Dysfunctions of Shank proteins have been reported in several neuropsychiatric disorders, i.e. autism, schizophrenia, and Alzheimer’s disease [102] (Table 3). Indeed, two de novo mutations of Shank3 have been associated with schizophrenia and schizoaffective disorder cases [103]. These mutations are predicted to affect Shank3 protein function. In particular, the R1117X mutation may cause Shank3 loss of function. Notably, in all the above cases, mental retardation was also diagnosed [103]. The Shank1 promoter variant rs3818280 has been associated with impaired working memory in schizophrenia, which depends on patients’ genotype (CC, CT, or TT). On the digit span task of the Wechsler Adult Intelligence Scale (WAIS-R), schizophrenia patients carrying either CT or TT genotypes have been shown to repeat less number of sequences in both forward and backward digit span subtests as compared to patients carrying CC genotype [104]. Similarly, in a population of at risk subjects for psychosis, subjects carrying the T allele performed worse than those carrying CC genotype in the forward digit span subtest [104]. Recently, different Shank1 gene deletions have been demonstrated in two unrelated ASD families from a population of European and Canadian individuals [105]. Several social-like impairments have been described in Shank1 (−/−) null mutant mice, including decreased levels of ultrasonic vocalizations and scentmarking behavior [106]. Mutations in the gene coding for Shank3 have been reported in ASD patients [67, 107–109]. Two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in ASD patients have been reported to affect spine development and morphology, as well as spontaneous neuronal activity in cultured neurons [110]. Notably, mice carrying Shank3 deletions (Shank3B −/− mice) exhibit self-injurious repetitive grooming behaviors and deficits in social interaction resembling autistic behaviors in humans

Mol Neurobiol Table 3 Clinical and preclinical evidence of Shank implication in the pathophysiology of psychotic disorders Experimental paradigm/ sample

Drug


Outcome

Reference

Shank3 mutants overexpression in cultured neurons

–

–

Immunocytochemistry, western blot, immunoprecipitation, electrophysiological recording

[110]

Shank1 (−/−) null mutant mice

–

–

Behavioral assessment

Shank3B −/− mice

–

–

Behavioral assessment, western blot, electrophysiological recording

Rat dissociated hippocampal neurons

Clozapine

In vitro 1.0 μM

Immunocytochemistry

Haloperidol In vitro 0.1 μM

Immunocytochemistry

Two Shank3 gene de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD differently affect dendritic spine development, morphology and neuronal activity Several social communication impairments, including decreased levels of ultrasonic vocalizations and scent-marking behavior Mice exhibited self-injurious repetitive grooming behaviors and deficits in social interaction resembling autistic behaviors; altered PSD composition in the striatum, with overall thinner and shorter PSDs and reduced spine density at medium spiny neurons Clozapine increased Shank1a protein density along primary and secondary dendrites, increased dendritic spine density in primary dendrites, increased the number of filopodia and mushroom dendritic spines Haloperidol decreased Shank1a protein density along secondary dendrites and decreased the number of filopodia in secondary dendrites Two de novo mutations (R1117X and R536W) associated with schizophrenia and schizoaffective disorder cases. Assays in zebrafish and rat hippocampal neurons revealed behavior and differentiation defects resulting from R1117X mutation Shank1 promoter variant rs3818280 was associated with impaired auditory working memory in schizophrenia, which depended on patients’ genotype (CC, CT, or TT) Subjects at risk for psychosis carrying a T allele performed worse than those carrying CC genotype in the forward digit span subtest Two different Shank1 gene deletions in a 4-generation family in which male carriers have ASD and in an unrelated ASD-affected patient

285 controls and 185 – patients with schizophrenia or schizoaffective disorder, with unaffected parents

–

Gene screening, variation analysis, assays in zebrafish and rat hippocampal neurons

199 schizophrenia patients, 206 healthy controls, 77 subjects at risk for psychosis

–

–

Genotyping of coding and promoter variants in Shank1, Shank2 and Shank3 genes; Wechsler Adult Intelligence Scale (WAIS-R) digit span test

1,158 Canadian and 456 European individuals with ASD

–

–

Genotyping

[180]

[111]

[24]

[103]

[104]

[62]

ACC anterior cingulate cortex; ASD autism spectrum disorders; cGMP cyclic guanosine monophosphate; dlCP, vlCP, dmCP, vmCP dorsolateral, ventrolateral, dorsomedial, and ventromedial caudate-putamen, respectively; IC insular cortex; ISHH in situ hybridization histochemistry; MAC medial agranular cortex; MC motor cortex; NO nitric oxide; PPI prepulse inhibition; RT-PCR reverse transcription polymerase chain reaction; SS somatosensory cortex

[111]. Shank3B −/− mice also showed reduced SAPAP3, Homer1b/c, and PSD93 protein levels and reduced GluR2, NR2A, and NR2B subunit levels in striatal PSD fractions.

These molecular alterations have been associated with overall thinner and smaller PSDs and with reduced spine density on medium-sized striatal spiny neurons in these mice [111].

Mol Neurobiol

Evidence exists that ASD-associated mutations in Shank3 may impair AMPAR and NMDAR signaling and may alter neurexin–neuroligin-mediated signaling in rat hippocampal neurons [112]. These findings may suggest that Shank protein aberrations could contribute to cognitive symptoms in schizophrenia and could be implicated in intellectual disability. Shank proteins appear critically involved in the regulation of dendritic spine morphology, architecture, and function. Despite being considered mere scaffolding molecules, Shank proteins are pivotal in glutamatergic signaling and their defects are prominently implicated in intellectual disability and autistic diseases. Impairment of activity-dependent synaptic plasticity has been suggested in these conditions [49], and Shank anomalies have been described to impair glutamate-mediated neurotransmission and dendritic spine morphology [41, 112].

of schizophrenia [120]. Mutant mice carrying a putative dominant-negative form of DISC1 (DN-DISC1) showed morphological, immunohistochemical, and behavioral abnormalities that may resemble schizophrenia alterations [121]. In the same model, several dopamine-related abnormalities, such as increased striatal D2R and DAT expression, decreased basal extracellular dopamine levels in ventral striatum, and higher dopamine increase after methamphetamine were found [122]. Moreover, in a transgenic mouse model with inducible expression of mutant human DISC1 restricted to forebrain regions, mild enlargement of the lateral ventricles, attenuation of neurite outgrowth in primary cortical neurons, reduced LIS1, SNAP-25 and endogenous DISC1 protein levels, and gender-dependent behavioral abnormalities were described [123].

DISC1

DISC1 in the Pathophysiology of Psychotic Disorders

DISC1 Regulates Multiple Intracellular Pathways in Neurogenesis and Neurodevelopment

DISC1 role in schizophrenia has been originally demonstrated in a large Scottish family sample, in which a balanced translocation of this gene originated directly disrupted transcripts that co-segregated with the disease [113]. Based on these findings, several animal models have been created reproducing DISC1 Scottish mutations [124]. Mutant DISC1 expression in mice has been shown to result in reduced serine racemase protein levels, thereby reducing D -serine production and leading to behavioral abnormalities consistent with a NMDAR hypofunction [121] (Table 4). The DISC1 genetic mouse model of schizophrenia also displays impaired synaptic connections between frontal cortical neurons [125]. Overall, the comprehensive analysis of the numerous DISC1 mutant mice revealed selective deficits in working memory and in neural circuits (i.e., prefrontal cortex, hippocampus) involved in the pathophysiology of schizophrenia and related mental disorders [126, 127]. Moreover, recent studies have demonstrated that DISC1 mutants display abnormal tangential migration of cortical interneurons during embryonal stages, as well as they have selective alterations in GABAergic neural subpopulations in cortex and hippocampus [128]. These findings are strikingly consistent with the aberrant interneurons laminar distribution patterns described in postmortem schizophrenia patients [129, 130]. These findings suggest that mutations of DISC1 may impair synaptic morphology and glutamate–dopamine signaling, with region-specific distribution. These suggestions have been confirmed in in vitro studies demonstrating that DISC1 mutations may directly affect axon and spine morphology in hippocampal and cortical neurons [131]. To confirm preclinical data on DISC1 role in schizophrenia pathophysiology, several DISC1 genetic variants have been associated with schizophrenia in different human populations [66, 132, 133].

Disrupted-in-schizophrenia 1 (DISC1 ) is a susceptibility gene for major mental disorders including schizophrenia, bipolar disorder, and major depression. DISC1 has been first characterized by cloning of a chromosomal translocation that segregated with a spectrum of major mental illnesses in a Scottish family [113, 114]. Besides the PSD, DISC1 has also been found in other subcellular localizations including centrosome, nucleus, cytoskeleton, growth cones, membranes, and mitochondria [115]. DISC1 is involved in several signaling pathways (including NMDAR-, GABA-, GSK3β-, and Wnt-mediated signaling pathways) and takes part in neurogenesis and neural development in adult brain [116]. DISC1 interacts with other proteins, such as neuregulin and dysbindin, along the Akt/ GSK signaling pathway, whose dysfunctions have been implicated in schizophrenia pathophysiology [13, 117, 118]. Indeed, aberrant interaction among these molecules may lead to NMDAR dysfunctions during neurodevelopment [13]. DISC1 has a major role during neurodevelopment, since it may regulate neuronal progenitor proliferation [119]. It may be hypothesized that these functions are probably exerted via modulation of GSK3beta/beta-catenin signaling, since DISC1 inhibits GSK3beta activity through a direct interaction [119] (Fig. 1). GSK3 inhibitors may normalize neural progenitor proliferation and schizophrenia-related behavioral abnormalities caused by DISC1 loss of function [119]. Recent studies have also highlighted the role of DISC1 in dopaminergic signaling via D1Rs [78], which may represent a further potential molecular crossroad between dopaminergic and glutamatergic dysfunctions in schizophrenia. Mice carrying DISC1 mutations are considered valuable animal models

Mol Neurobiol Table 4 Clinical and preclinical evidence of DISC1 modulation by dopaminergic and glutamatergic stimuli and of its implication in the pathophysiology of psychotic disorders Experimental paradigm/sample

Drug


DN-DISC1 transgenic mice (mice carrying a dominant-negative form of DISC1, DN-DISC1, which is expressed under the CaMKII promoter)

–

–

–

–

Transgenic mice model of inducible – expression of mutant human DISC1 restricted in forebrain areas

–

Pyramidal neurons from medial prefrontal cortex in two mouse models: mDISC1 mice (expressing a truncated mouse DISC1 protein throughout the entire brain) and hDISC1 mice (expressing a truncated human DISC1 protein in forebrain regions

–

–

Acute adm. in L100P DISC1 mutant mice

Haloperidol 0.4 mg/kg i.p. Clozapine 3 mg/kg i.p.

Repeated adm. in polyI:C/DNDISC1 transgenic mice

Haloperidol 1 mg/kg once a day oral Clozapine 3 mg/kg once a day oral

21 days adm. in mice

Haloperidol 0.05 mg/kg i.p. Olanzapine 0.04 mg/kg i.p. Risperidone 0.025 mg/kg i.p. Clozapine 0.5 mg/kg i.p. –

–

RT-PCR, in situ hybridization, in vivo magnetic resonance imaging, immunohistochemistry, behavioral assessment

Outcome

DN-DISC1 transgenic mice show lateral ventricles enlargement, decreased parvalbumin immunoreactivity in the cortex, and several behavioral impairments, including locomotor hyperactivity, altered sensorimotor gating, altered olfactory-associated behavior, and increased immobility in the forced swim test Several dopamine-related PET scan with [11C]raclopride, autoradiography of D2Rs with abnormalities, such as increased [3H]-Spiperone, real-time striatal D2R and DAT expression, PCR, open field and in vivo decreased basal extracellular microdialysis after dopamine levels in ventral methamphetamine, striatum, and higher dopamine immunoblot for DAT increase after methamphetamine (1 mg/kg i.p.) Western blot, Mutant human DISC1 transgenic immunoprecipitation, mice show enlargement of lateral histopathological and ventricles, attenuation of neurite immunohistochemical assays, outgrowth in primary cortical magnetic resonance imaging, neurons, reduced LIS1, SNAPbehavioral assessment 25, and endogenous DISC1 protein levels, gender-dependent behavioral abnormalities Whole-cell patch clamp In cortical pyramidal neurons from recordings both models the frequency of spontaneous EPSCs is increased. Male mice are more affected in both models, exhibiting increases in the ratio of excitatory to inhibitory events. Sex-specific changes in spontaneous IPSCs are observed in the mDISC1 model Behavioral assessment Partial amelioration in PPI deficits Behavioral assessment Partial amelioration in PPI deficits clozapine abolishes the disruption of latent inhibition in 100P/100P mice Behavioral assessment Suppression of increased MK-801induced hyperactivity Behavioral assessment Amelioration of cognitive impairment suppression of increased MK-801-induced hyperactivity Real-time quantitative RT-PCR No changes in DISC1 expression in (DISC1 mRNA) frontal cortex and hippocampus Real-time quantitative RT-PCR Increased DISC1 expression in frontal cortex and hippocampus (DISC1 mRNA) Real-time quantitative RT-PCR Increased DISC1 expression in (DISC1 mRNA) frontal cortex Real-time quantitative RT-PCR No changes in DISC1 expression in (DISC1 mRNA) frontal cortex and hippocampus

Reference

[121]

[181]

[123]

[125]

[81]

[162]

[80]

[113]

Mol Neurobiol Table 4 (continued) Experimental paradigm/sample

Drug


A large Scottish family carrying a balanced (1;11)(q42.1;q14.3) translocation which segregates with schizophrenia and related psychiatric disorders –

–

Case–control study; a set of 222 – French Caucasian schizophrenic patients and 151 healthy unrelated controls

–

–

–

A set of Chinese Han individuals, including 310 schizophrenic patients and 400 controls

A Japanese sample of 33 schizophrenia patients and 29 healthy comparison subjects

Outcome

Reference

DNA sequencing, PCR, northern The (1;11)(q42.1;q14.3) blot analysis translocation disrupts two novel genes, named DISC1 and DISC2, suggesting these genes may be considered candidate genes for susceptibility to psychiatric illness Genotyping Three short tandem repeat loci are [66] associated with schizophrenia: (ATCC)n1, D1S1621, and (ATCC)n2. The short tandem repeats occur in intronic sequences near to a critical splice junction that gives rise to the expression of DISC1 isoforms Psychopathological assessment The DISC1 rs3738401 missense [132] (DIGS, BPRS-E, CGI-S, GAF, variant is significantly more PANSS), genotyping frequent in ultra-resistance to antipsychotic treatment than in treatment-responding schizophrenia patients, suggesting that DISC1 variants may have a functional influence on response to antipsychotic treatment Magnetic resonance imaging, The DISC1 Ser704Cys [133] genotyping polymorphism may be relevant to medication effect on brain morphology in treated schizophrenia patients: In Ser homozygote patients, the right medial superior frontal gyrus volume is correlated with the daily dose of antipsychotic medication

BPRS-E expanded version of the Brief Psychiatric Rating Scale; CaMKII calcium/calmodulin-dependent protein kinase II; CGI-S Clinical Global Impression-Severity Scale; D2R D2 dopamine receptor; DAT dopamine transporter; DIGS Diagnosis Interview for Genetic Studies; DN-DISC1 dominant-negative form of DISC1; EPSC excitatory postsynaptic currents; GAF Global Assessment of Functioning Scale; hDISC1 human DISC1; IPSC inhibitory postsynaptic currents; LIS1 lissencephaly-1; mDISC mouse DISC1; PANSS Positive and Negative Syndrome Scale; PCR polymerase chain reaction; polyI:C/DN-DISC1 transgenic mice polyriboinosinic–polyribocytidylic acid (polyI:C)-treated DN-DISC1 transgenic mice, a mouse model of mental disorders obtained by inducing abnormal immune response during the perinatal period in mice with overexpression of the human dominant-negative form of DISC1; PPI prepulse inhibition; RT-PCR reverse transcription polymerase chain reaction; SNAP-25 synaptosomalassociated protein 25

Other Proteins Several other PSD and PSD-related proteins have been found impaired in preclinical and clinical studies on schizophrenia. MAGUKs The MAGUKs are a superfamily of PSD-95-related scaffolding proteins comprising SAP-102, PSD-93, or SAP-97.

SAP-102 mRNA expression has been found increased in hippocampal tissue after rat isolation rearing [134], which is considered a valid neurodevelopmental model of schizophrenia [135, 136]. Expression of SAP-102 has also been found decreased in striatum of schizophrenia patients [137], whereas gene expression in thalamus has been reported differentially expressed in the brain of schizophrenia patients based on the age of the subjects: decreased in young schizophrenia patients [13] and increased in older ones [14].

Mol Neurobiol

Increased PSD-93 transcript, but decreased protein, has been reported in the anterior cingulate cortex of schizophrenia patients [50], showing pattern of expression similar to PSD95 in this region. A decreased protein expression of SAP-97 has been shown in the PFC of schizophrenia patients [138]. Notably, SAP-97 transcripts have been found upregulated in rat adult neocortex after acute injection of the NMDAR noncompetitive antagonists phencyclidine and dizocilpine, but not after administration of the indirect dopamine agonists cocaine and methamphetamine [139].

Kalirin Kalirin is a GDP/GTP exchange factor (GEF), which interacts with PSD-95 and spinophilin through its C-terminus PDZbinding motif (Fig. 1). Kalirin is an essential protein in mature excitatory synapses implicated in spine and synapse formation [140]. KALRN KO mice show several structural, functional, and behavioral alterations inherent to schizophrenia pathophysiology, including (a) decreased cortical, but not hippocampal, Rac1 activity; (b) decreased cortical, but not hippocampal spines density; (c) reduced cortical AMPAR currents; (d) impaired working memory and sociability; (e) reduced prepulse inhibition response; and (f) locomotor hyperactivity, reversed by clozapine [20]. Kalirin knockdown by RNA interference in cultured neurons has been shown to affect dendrite morphology [141]. Decreased spine density, impaired activity-dependent spine plasticity, and decreased complexity of dendritic trees have been reported in cortical pyramidal neurons of KALRN-null mice [141]. Kalirin has been implicated in different neuropsychiatric diseases, above all in schizophrenia. Multiple missense rare mutations in KALRN gene have been proposed as a risk factor for schizophrenia [142]. Furthermore, decreased Duo (the human ortholog of the murine Kalirin-7) mRNA levels and decreased spine density have been found in PFC of schizophrenia patients [143]. Therefore, although still scarce, the above findings suggest that kalirin may represent a promising candidate in the research on schizophrenia and behavioral disorders pathophysiology. Other preclinical and clinical studies have reported alterations in a number of other PSD-related proteins, such as neurofilament-light [13, 14, 50], SAPAP [144], or caldendrin [145]. However, the functional role and biological relevance of these alterations are yet to be determined. Overall, the molecular changes of PSD and PSD-related proteins discussed herein suggest a defect in PSD functioning in schizophrenia. Abnormal PSD functioning may cause glutamate dysfunctions and aberrant interplay with other neurotransmitter systems relevant to schizophrenia pathophysiology, such as the dopaminergic and the serotonergic ones [146, 147].

PSD Protein Modulation by Antipsychotic Drugs Antipsychotic therapies impact mainly, albeit not exclusively, dopaminergic neurotransmission, since all available antipsychotics act as antagonists at dopamine D2 receptors, although with different degrees [148]. Notably, a large part of antipsychotics also interact with other dopamine receptors, besides D2Rs [149]. However, evidence is accumulating that antipsychotic drugs may achieve part of their effects by inducing long-term adaptive changes at glutamatergic postsynaptic sites in brain regions relevant to psychosis, such as the PFC, the striatum, and the hippocampus [46, 147, 150]. A role in antipsychotic drug action has been postulated for PSD scaffolding proteins, which are master organizers of postsynaptic Ca2+ networks contributing to drug-induced neuroplastic changes [70]. In the next paragraphs, we will discuss the most recent findings regarding the involvement of PSD scaffolding proteins in the mechanisms of action of typical and atypical antipsychotics, as well as their possible role as molecular targets for future antipsychotic strategies. PSD-95 Preclinical studies have shown that PSD-95 gene expression is modulated by both acute and chronic antipsychotic treatments, with a region-specific pattern and with effects dependent on treatment duration (Table 1). A recent report has described a decrease in PSD-95 expression in selected subregions of the rat cortex following acute haloperidol or quetiapine administration [20]. Conversely, PSD-95 expression has been found significantly increased in cortical regions by acute co-administration of haloperidol and valproate [20]. Unlike cortical regions, in striatum, no significant changes in PSD-95 expression have been recognized in acute paradigms [16, 22]. On the other hand, a 21-days chronic treatment by either typical or atypical antipsychotics has been reported to increase PSD-95 expression in this region [16, 22]. Therefore, PSD-95 modulation by chronic antipsychotics may underlie specific synaptic plasticity changes, potentially related to prolonged treatments and affecting glutamatergic signaling in striatum. Provided the suppressive effects of PSD95 on D1R-mediated signaling [35, 36], the increase in PSD95 expression may contribute to slow down D1R-mediated overstimulation, which in turn results from prolonged D2R blockade by long-term antipsychotic treatments. It has been suggested that PSD-95 may be necessary for atypical antipsychotic action at serotonin receptors [46]. PSD95 may participate in the clozapine molecular mechanisms of action in mice, since PSD-95 null mice do not show the clozapine-mediated normalization of PCP-disrupted prepulse inhibition [46]. These effects are thought to involve 5-HT2A receptors, since in PSD-95 null mice, the selective antagonists

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at 5-HT2A receptors, such as M100907 and SR46349B, failed to reverse PCP-disrupted prepulse inhibition [46]. The putative role of PSD-95 in the modulation of serotonergic signaling has been also confirmed by the observation that the selective serotonin reuptake inhibitor antidepressants significantly increased PSD-95 expression in rat cortex and striatum [20]. Thus, PSD-95 may represent a valuable adaptor molecule that cross-links glutamatergic, dopaminergic, and serotonergic transmission, thus providing a postsynaptic modulation of these systems. This particular action of PSD-95 may deserve specific attention for future antipsychotic strategies that would overcome the receptor level and would directly target the postsynaptic signaling compartment. Besides its modulation by current antipsychotic treatments, recent microRNA studies have indicated the possibility of using PSD-95 transcriptional regulation as a potential therapeutic strategy for the treatment of neuropsychiatric disorders. MiR-485 has been demonstrated to negatively regulate spines density, PSD-95 clustering, and surface GluR2 expression in cultured hippocampal neurons, thus providing new approaches to synaptic plasticity dysfunctions in severe neurological diseases, such as Huntington’s or Alzheimer’s disease [120]. Notably, PSD-95 translation may be bidirectionally regulated by mGluR-mediated signaling through the control of phosphorylation status of both the fragile X mental retardation protein and the microRNA miR-125a [151]. Homer Among Homer genes, the inducible immediate-early gene isoform Homer1a is known to respond to dopaminergic manipulations [152]. A number of preclinical studies have shown that Homer1a expression may be induced by acute antipsychotic administration in brain regions relevant to schizophrenia pathophysiology and may be differently modulated by typical and atypical antipsychotics, potentially according to their dopaminergic receptor profile (Table 2). The high D2Rblocking antipsychotic haloperidol has been shown to induce Homer1a in all striatal subregions, with prominent impact on the dorsal and lateral regions, and in both the core and the shell of the nucleus accumbens [18, 150, 153]. Another antipsychotic with high D2R affinity, (−)-sulpiride, may induce Homer1a in the ventrolateral subregion of the caudateputamen and in the core of the accumbens [21]. Differently from typical antipsychotics, atypical antipsychotics have been demonstrated to induce a Homer1a region-specific expression depending also on their affinity to receptors other than the dopaminergic ones, such as the serotonergic receptors, as well as on the dose administered. Risperidone, which has elevated D2R affinity but lower maximal binding effect compared to haloperidol [106], may induce Homer1a expression in the lateral regions of the caudate-putamen only [21].

Olanzapine-induced expression of Homer1a gene has been reported in the core of the nucleus accumbens only [150], although high doses of the compound may also elicit Homer1a expression in the lateral regions of the caudateputamen [21]. Ziprasidone, a high D2R affinity atypical antipsychotic, differentially impacts Homer1a expression, according to dosage. At low doses, ziprasidone may produce a striatum-specific Homer1a induction, while a wider gene induction, spreading also to the cortex and the nucleus accumbens, has been found at high doses, probably depending on higher involvement of serotonergic receptors [16]. Moreover, the expression of Homer1a in the striatum by ziprasidone is significantly higher at high doses, which are correlated to the liability to extrapyramidal side effects in animal models [154]. Also, the dopamine partial agonist aripiprazole has been demonstrated to differentially modulate Homer1a expression depending on the dose. A strong induction of Homer1a expression has been observed in all caudateputamen subregions by acute administration of low aripiprazole doses [18]. High aripiprazole doses have no significant effects in the striatum but may induce Homer1a expression in the cingulate cortex and in the inner and outer layers of the frontal cortex [18]. These dose-dependent effects may be likely due to aripiprazole mixed agonist/antagonist activity at pre- and postsynaptic D2Rs. Basically, low aripiprazole doses are supposed to exert a prevalent antagonist activity at postsynaptic D2Rs, thus directly inducing Homer1a striatal expression [155], whereas high doses may exert a prevalent agonist activity at presynaptic D2 autoreceptors, thereby having scarce effects on Homer1a expression [18]. Atypical antipsychotics with low D2R affinity, such as clozapine, or with unique D2R dissociation kinetics, such as quetiapine and sertindole, have been observed to modulate Homer1a expression with specific patterns in striatum. Indeed, clozapine has been shown to acutely induce Homer1a expression in the nucleus accumbens only [16, 18]. Acute clozapine may also induce Homer1a expression in the cortex (in the anterior cingulate, medial agranular, somatosensory and insular cortices), probably due to its impact on serotonergic receptors [16, 18]. Quetiapine, a fast dissociating D2R antagonist, has been found to slightly induce Homer1a striatal expression [153]. However, quetiapine may induce a robust Homer1a expression in the cortex when acutely administered, whereas significantly decreasing it in chronic paradigms [20]. This feature may strengthen the hypothesis of a combined dopaminergic–serotonergic control of Homer1a expression in the cortex. Indeed, acute administration of the serotonergic-selective antipsychotic sertindole has been found to reduce Homer1a expression in the somatosensory and insular cortices [22]. Taken together, these lines of evidence support the view that Homer1a could represent a molecular sensor of glutamatergic postsynaptic involvement in the mechanism of action of antipsychotics. Moreover, since the above-mentioned findings suggest

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that Homer1a induction by antipsychotics may be related to their propensity to perturb dopamine transmission, the pattern of Homer1a expression may be considered as a predictor of the liability of each antipsychotic to induce extrapyramidal side effects [153]. Homer1a has been demonstrated to preserve its expression profile in the striatum after chronic antipsychotic administration, and it seems to be unaffected by the tolerance or desensitization phenomena observed for other immediate-early genes, such as c-fos [156, 157]. Indeed, haloperidol has been demonstrated to modulate Homer1a expression with similar patterns in both acute and chronic paradigms [18, 22]. Nonetheless, some adaptive changes should not be ruled out after chronic antipsychotic administration. Chronic clozapine treatment, as opposed to acute treatment, has been reported to produce no significant Homer1a changes in the caudateputamen, whereas it may decrease Homer1a expression in the cingulate cortex and in the inner layers of both frontal and parietal cortices. In opposition to what observed after acute treatment, chronic ziprasidone may induce Homer1a expression only in the lateral regions of the caudate-putamen and in the core of the nucleus accumbens [16]. Lastly, chronic aripiprazole, differently from acute administration, has been demonstrated to induce Homer1a in the lateral regions of the caudateputamen while reducing it in the cingulate and in the inner layers of the frontal cortex [18]. These effects might be part of an adaptive response of the glutamatergic system to chronic antipsychotic treatment [18]. Overall, despite Homer1a modulation by antipsychotics appears to be not susceptible of tolerance, acute or chronic treatment may result in different patterns of gene expression, probably accounting for neuroplastic adaptations triggered by prolonged treatments. Besides the inducible isoform Homer1a , dopaminergic drugs have also been demonstrated to modulate the expression of the constitutive isoforms of Homer1 gene, thus putatively inducing direct rearrangements in synaptic architecture. The acute administration of selective antagonists at D2Rs and D4Rs, as well as of a D2R partial agonist (i.e., terguride), has been demonstrated to reduce Homer1b/c expression in the striatum and in cortical subregions [155]. Nevertheless, the D1R selective antagonist SCH-23390 has been described to increase Homer1b/c expression in the core of the nucleus accumbens, while reducing it in the motor cortex [155]. However, the evaluation of Homer1b/c modulation after acute or chronic antipsychotic administration has provided contrasting results. Acute haloperidol treatment, indeed, has been observed to reduce Homer1b/c expression in the ventrolateral caudate-putamen, in the shell of the nucleus accumbens, and in the motor, somatosensory, and insular cortices [155], whereas no significant differences in expression have been found after a 16-day chronic treatment by haloperidol or quetiapine [16]. However, treatment duration may be a critical

factor to cause changes in Homer1b/c expression, which has been found to increase in both striatum and cortex following a 21-day haloperidol or sertindole treatment [22]. A recent work has also demonstrated that chronic treatment with the mood stabilizers lithium or valproate—which are widely used as therapeutic add-on strategies to antipsychotics in schizophrenia or in bipolar disorder—may directly impact the expression of Homer1b/c and related synaptic genes (Shank and inositol 1,4,5 trisphosphate receptors) in cortical and subcortical regions [147]. Moreover, valproate add-on to either haloperidol or quetiapine has been reported to elicit changes in Homer1a expression patterns different from those induced by either drugs when given alone [20]. These findings confirm the role of Homer1 genes in synaptic rearrangements triggered by antipsychotic treatments. Notably, a recent clinical study has reported that two Homer1 polymorphisms may be associated with clinical response (expressed as improvement on PANSS subscores) in schizophrenia patients after different 4 week antipsychotic treatments [89]. Taken together, clinical and preclinical data suggest that Homer1 genes might be crucial in the pathogenesis and in the severity of psychotic symptoms, as well as in determining the efficacy of antipsychotic drugs. Shank Several studies have demonstrated that typical and atypical antipsychotics may differentially impact dendritic spine formation and synaptogenesis through the direct rearrangement of synaptic architecture, e.g. by modulating postsynaptic scaffolding proteins, in particular Shank (Table 3). Clozapine and haloperidol may specifically affect Shank protein density in dendritic spines. In rat dissociated hippocampal neurons, in vitro clozapine administration has been found to increase Shank1a protein density along dendrites, whereas haloperidol caused a decrease in Shank1a density [24]. The observed increase may be explained as a possible specific increase in shaft synapses or as the formation of multiple synapses per dendritic spine. The different modulation of Shank expression by clozapine and haloperidol may be consistent with a different impact on dendritic spine formation by the two antipsychotics. Clozapine, indeed, but not haloperidol, has been reported to increase dendritic spine density in primary dendrites and the number of filopodia in secondary dendrites and to up-regulate dendritic spines [24]. These effects have been suggested to contribute, at least in part, to higher efficacy of clozapine in treatment-resistant schizophrenia [158]. DISC1 As described above, DISC1 is a multifunctional scaffolding protein that interacts with different proteins involved in

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neuronal migration, neurite outgrowth, cytoskeletal modulation, and signal transduction [159]. DISC1 participates in the maintenance of spine morphology and function by anchoring Kalirin-7, a GEF for Rac1, and regulating the access of Kalirin-7 to Rac1, a step that is crucial for controlling Rac1 activation in response to NMDAR stimulation [160] (Fig. 1). DISC1 may also interact with the Traf2 and Nck-interacting kinase, stabilizing the levels of postsynaptic density proteins to regulate synaptic composition and activity [161]. Chronic administration of the atypical antipsychotics olanzapine and risperidone at clinically relevant doses has been shown to increase DISC1 mRNA expression in mice frontal cortex, whereas haloperidol and clozapine have no effect. Olanzapine may increase DISC1 mRNA expression also in the hippocampus and a similar trend has been shown for risperidone [80]. No data exist at present that may help to understand these differences in DISC1 gene modulation by specific antipsychotics. Moreover, no studies have been performed measuring DISC1 protein modulation by different antipsychotics. It is possible that these differences may derive from the specific receptor affinity profile of each antipsychotic. However, these results suggest that DISC1 may be implicated in antipsychotic mechanisms of action (Table 4), although additional studies are needed to dissect if its expression changes may be specific for drug and for brain region. The increase in DISC1 expression by chronic atypical antipsychotic treatment may suggest a putative impact on DISC1mediated modulation of synaptic spines as a potential mechanism of action of these agents. Repeated clozapine, but not haloperidol, oral administration has been reported to improve cognitive impairment in the polyI:C-treated DN-DISC1 (polyI:C/DN-DISC1) transgenic mice [162], a mouse model of behavioral disorder obtained by inducing abnormal immune response during the perinatal period in mice through the overexpression of the human DN-DISC1 . In this animal model, both clozapine and haloperidol suppress the MK801-induced hyperactivity, whereas no effects on social behavior impairments have been observed with both antipsychotics [162]. In clinical studies, DISC1 missense variants have been associated to schizophrenia patients who display ultraresistance to treatments, defined as patients who continue to experience positive psychotic symptoms despite a period of clozapine therapy of at least 6 weeks and at least two unsuccessful previous trials with conventional or atypical antipsychotic drugs [132]. These results suggest that DISC1 variants may have a functional influence on the response to antipsychotic treatment. Indeed, a DISC1 Ser704Cys polymorphism has been shown to affect the effects of therapies on brain morphology in treated schizophrenia patients [133]. Patients carrying the Cys allele variant show smaller grey matter volume in supramarginal gyrus than Ser carriers [133]. Ser homozygotes show a direct correlation between the right

medial superior frontal gyrus volume and the daily dose (in haloperidol equivalents) of antipsychotic medication, thus implicating a direct role of DISC1 in the effects of antipsychotics on brain morphology [133]. Overall, clinical and preclinical data suggest that DISC1 may represent an interesting postsynaptic molecule mediating antipsychotic-induced cytoarchitectural plasticity.

Discussion Understanding protein–protein interactions at PSD could help to clarify the role of PSD proteins in normal and pathological synaptic plasticity, as well as in response to pharmacological challenges by psychotropic drugs. Scaffolding PSD proteins may modulate dendritic spines morphology and function in several ways. They contribute to regulate trafficking and localization of multiple proteins in spine microdomains, to bridge receptors to their intracellular effectors along second messenger pathways, and to allow cross-talk among different neurotransmitter systems [7, 9, 146, 163]. Through this complex range of biological actions, PSD proteins may participate to activity-dependent long-term synaptic changes, which comprise both enduring morphological rearrangements (e.g. spine formation, enlargement, or shrinking) and sustained changes in synaptic signaling strength (i.e., LTP or LTD [9]). PSD proteins may be considered valuable candidates in the pathophysiology of synaptic diseases, such as schizophrenia and schizophrenia-related disorders, and may represent intriguing targets for future therapeutic approaches. Since the PSD is primarily implicated in glutamatergic signaling and glutamate-mediated activity-dependent synaptic plasticity, abnormal PSD functioning may crucially contribute to glutamatergic dysfunctions and “synaptopathies” that have been hypothesized in schizophrenia [164]. PSD also represents a site of integration and cross-talk between the glutamatergic and other neurotransmitter systems relevant to schizophrenia pathophysiology, such as the dopaminergic and the serotonergic ones. Therefore, aberrant PSD functioning may also underlie dysfunctions in dopamine–glutamate–serotonin cross-talk [5, 165]. It appears that loss of function or lack of some PSD proteins, such as PSD-95, Homer, or Shank, may impair glutamatergic signaling, in turn disrupting activity-dependent synaptic plasticity as well as dendritic spine architecture and morphology. Although more studies need to be carried out, some molecular mechanisms putatively implicating PSD in the pathophysiology of schizophrenia and behavioral disorders, such as ASD, may be hypothesized. The major function of PSD-95 proteins is to connect NMDARs to their intracellular effectors [30, 166]; therefore, aberrant PSD-95 may strongly affect glutamatergic transmission NMDAR mediated. Dysfunctions of PSD-95 proteins may also increase surface D1Rs (due to disruption of the

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PSD-95-mediated D1R internalization) [36], as well as influence AMPAR signaling [166], and impair 5-HT2A/5-HT2C receptor trafficking [43]. These abnormal neurotransmitter interactions might lead to a multireceptor impairment, which may result in reduced glutamatergic signaling and increased D1R transmission and aberrant serotonergic signaling in postsynaptic neurons. Moreover, PSD-95 has been demonstrated to recruit other PSD components to regulate dendritic spine architecture. The overexpression of PSD-95 not only terminates dendritic spine enlargement, but also promotes generation of multi-innervated spines [33]. Therefore, PSD-95 dysfunctions may cause synaptic shrinkage and may affect longterm changes in synaptic strength, thereby impairing synaptic plasticity. On the other hand, the excessive intracellular signaling via the PSD-95/NOS pathway has been considered detrimental, thus predisposing to glutamatergic hyperexcitability in some clinical conditions, e.g. stroke [167]. The different isoforms of Homer proteins are in reciprocal and functional balance. Basically, a balanced ratio between long and short Homer proteins may preserve postsynaptic neuron potentials and homeostatic scaling [94, 95], as well as glutamatergic signaling [66, 168], dopamine–glutamate interplay [21], surface ion channels opening and functioning [69], and dendritic spines architecture [72, 95, 169]. Impairment of this balance, such as by genetic manipulations, have been reported to cause relevant neurochemical, structural, and behavioral alterations [30, 85, 87]. The loss or reduction of functional long Homer proteins may depend on either genetic causes or overexpression of short Homer isoforms, such as Homer1a. Reduction of Homer1b/c expression or increase of Homer1a expression may disrupt Homer1b/c-mediated clusters that connect glutamate receptors to their intracellular effectors [168], thereby modifying local ionic concentrations (due to altered Ca 2+ release from internal stores and ion flow from surface ion channels) [70], and affecting synaptic architecture, since Homer proteins bridge PSD components to cytoskeleton [95]. Moreover, reduction of Homer1b/c expression or excessive Homer1a induction may ultimately lead to downscaling of local potentials [94], thus putatively causing a reduced postsynaptic neuron activation, which is at least in part functionally equivalent to glutamatergic hypofunction. Shank proteins have been implicated in dendritic spines maturation and enlargement as well as in the regulation of the synaptic signaling strength. Transfection of the Shank3 isoform in cerebellar granule cells has been found to induce the formation of new synapses and to increase NMDAR- and AMPAR-mediated currents [101]. It has also been reported that the Shank3 isoform elicits transynaptic changes in presynaptic protein levels and function, thereby modulating synaptic transmission at excitatory, glutamatergic synapses [41, 112]. Expression in cultured neurons of Shank isoforms

carrying ASD-associated mutations has been found to interfere with excitatory signaling, in particular reducing AMPA and NMDA receptor-mediated excitatory postsynaptic currents [41, 112]. On the light of these observations, it could be hypothesized that dysfunctions in Shank proteins may cause reduced glutamatergic signaling and may lead to severe morphological aberrations in dendritic spines. Alterations of other PSD and PSD-related proteins have been less studied; however, considered their physiological role it is attempting to hypothesize that their dysfunctions could also affect glutamatergic signaling, synaptic plasticity, and dendritic spines morphology. Further studies in this field are warranted. Despite the above-mentioned observations, in several studies the expression of PSD genes has been found increased, rather than decreased, in schizophrenia patients [50]. Several explanations can be proposed for this finding. For example, methodological bias, such as the homogeneity of patients’ sample selection in the case of postmortem studies (i.e. “mixed” schizophrenia diagnosis, such as schizoaffective disorder, or not well defined pre-mortem antipsychotic treatment), may account for these results. Also, PSD protein gene expression may be increased as a compensatory mechanism to preserve physiological PSD functioning. Nonetheless, it cannot be excluded that molecular mechanisms leading to schizophrenia may include excessive PSD protein activity (e.g. glutamatergic excitotoxicity) [170] and/or the formation of non-functional synapses that might impair cerebral connectivity [59]. Despite the fact that PSD proteins functions have been mostly studied in striatal tissue of adult rats, it can be hypothesized that PSD proteins may exert different molecular actions depending on the brain region, neuronal type (i.e., striatal neurons vs. cortical neurons), and neurodevelopmental stage. Should this be the case, it is to be expected that PSD protein dysfunctions may be different and lead to different consequences based on the brain region and the age they occur. Recent studies have demonstrated that currently available antipsychotics may modulate gene expression of some PSD proteins. Both typical and atypical antipsychotics have been reported to induce Homer1a expression in striatum, either after acute or chronic administration [16, 150, 153, 171]. Transient Homer1a induction by antipsychotics may be a mechanism to reduce postsynaptic hyperactivation consequent to stimulation of D1R by residual synaptic dopamine. Also, Homer1a induction may directly depend, at least in part, upon the blockade of postsynaptic D2Rs by antipsychotics. Therefore, Homer1a induction may be a sensible marker of dopamine perturbation by antipsychotics. Consistent with this view, Homer1a may be differently induced by antipsychotics according to their dopaminergic profile. Indeed, higher Homer1a induction has been observed by antipsychotics displaying a higher degree of D 2 R blockade [21]. Furthermore, antipsychotics have also been found to acutely

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induce other short Homer isoforms, such as Ania-3 [18, 153], or other inducible PSD-related genes, such as Arc [19, 172]. Constitutive PSD genes, such as Homer1b and PSD-95, have been poorly affected in acute paradigms of antipsychotic administration [155], whereas significant changes have been observed after prolonged antipsychotic treatments. Expression of both PSD-95 and Homer1b genes has been found increased after chronic haloperidol treatment, whereas ziprasidone only mildly increased gene expression, and sertindole had no effects [16, 22]. These findings may suggest that at least some antipsychotics may exert their chronic effects by modulating PSD protein functions. Antipsychoticmediated increase of these molecules may determine longterm improvements of glutamatergic signaling, as well as of dopamine–glutamate–serotonin interplay, thus contributing to restore synaptic plasticity processes, and dendritic spine morphology. These suggestions could possibly open new avenues of investigation for schizophrenia treatment. Current therapies for schizophrenia are jeopardized by high rates of poor response, in part as a result of the lack of really true innovative mechanisms of action among available antipsychotics [173]. Novel therapeutic approaches are therefore mandatory to overcome, at least in part, the drawbacks of schizophrenia therapy. One possible novel therapeutic target may be represented by the modulation of PSD proteins discussed herein, as well as other PSD proteins that have been less characterized since now for their involvement in schizophrenia, such as GRIP, SAP102, and Preso1. Targeting PSD proteins to unveil novel treatments for behavioral disorders should be balanced by the observation that mutations in the gene sequence of these proteins have been linked to pathophysiology of intellectual disability and autism-like phenotypes. Therefore, it will be fundamental to consider this limitation and evaluate consequently the strategy to undertake in designing new therapeutics possibly acting at the PSD. A recent pharmaceutical strategy has pointed on the generation of cell permeable peptides (CPPs), i.e., small peptides that may cross the blood–brain barrier and pass through neuron surface membrane [174]. Small inhibitory peptides are CPPs that link scaffolding proteins, such as PSD-95, by protein–protein interaction. By this mechanism, small inhibitor peptides prevent scaffolding proteins to interact with their targets [174]. An anti-PSD-95 small inhibitory peptide is currently under evaluation, also in humans, in stroke, to reduce the PSD-95/NOS pathway stimulation consequent to cortical ischemic damage [175]. The possibility to use small inhibitory peptides also in schizophrenia (e.g. to block Homer1a interaction with Homer1b) has not been tested to date. However, it may represent an intriguing strategy for future researches. Another putative target could be the inhibition of PSD-95/D1R interaction selectively in the cortex, in order to increase dopamine signaling in this region,

since some symptoms of schizophrenia have been attributed to low cortical D1R-mediated signaling [176]. Taken together, these observations indicate that PSD proteins are crucial molecules in synaptic architecture and function, involved in neuropsychiatric disorders pathophysiology, mostly in schizophrenia and related diseases, with the possibility to be targeted, directly or indirectly, by novel treatment strategies. Finally, considering the present lack of specific pharmacological treatments for autism and the deep involvement of PSD proteins in autism spectrum disorders, further studies exploring PSD proteins as potential targets in these classes of child diseases are warranted. Acknowledgments All members of the Laboratory of Molecular and Translational Psychiatry contributed to ideas and comments on the present work. Conflict of Interest Andrea de Bartolomeis has received unrestricted research funding from Astra Zeneca, Janssen-Cilag, and Lundbeck. The funding was made available to the Department of Neuroscience, University of Naples Federico II. He has received honoraria as speaker at educational activity sponsored by Astra-Zeneca Italia, Janssen-Cilag Italy, Eli Lilly, and Bristol-Myers Squibb. All other authors declare that, except for income received from our primary employer, no financial support or compensation has been received from any individual or corporate entity over the past 3 years for research or professional service, and there are no personal financial holdings that could be perceived as constituting a potential conflict of interest.

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