Hyperfunction of post-synaptic density protein 95 promotes seizure response in early-stage aß pathology.

Accumulation of amyloid-beta (Aß) can lead to the formation of aggregates that contribute to neurodegeneration in Alzheimer's disease (AD). Despite globally reduced neural activity during AD onset, recent studies have suggested that Aß induces hyperexcitability and seizure-like activity during the early stages of the disease that ultimately exacerbate cognitive decline. However, the underlying mechanism is unknown. Here, we reveal an Aß-induced elevation of postsynaptic density protein 95 (PSD-95) in cultured neurons in vitro and in an in vivo AD model using APP/PS1 mice at 8 weeks of age. Elevation of PSD-95 occurs as a result of reduced ubiquitination caused by Akt-dependent phosphorylation of E3 ubiquitin ligase murine-double-minute 2 (Mdm2). The elevation of PSD-95 is consistent with the facilitation of excitatory synapses and the surface expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors induced by Aß. Inhibition of PSD-95 corrects these Aß-induced synaptic defects and reduces seizure activity in APP/PS1 mice. Our results demonstrate a mechanism underlying elevated seizure activity during early-stage Aß pathology and suggest that PSD-95 could be an early biomarker and novel therapeutic target for AD.

Neonatal Isoflurane Anesthesia or Disruption of Postsynaptic Density-95 Protein Interactions Change Dendritic Spine Densities and Cognitive Function in Juvenile Mice.

BACKGROUND: Experimental evidence shows postnatal exposure to anesthesia negatively affects brain development. The PDZ2 domain, mediating protein-protein interactions of the postsynaptic density-95 protein, serves as a molecular target for several inhaled anesthetics. The authors hypothesized that early postnatal disruption of postsynaptic density-95 PDZ2 domain interactions has persistent effects on dendritic spines and cognitive function. METHODS: One-week-old mice were exposed to 1.5% isoflurane for 4 h or injected with 8 mg/kg active postsynaptic density-95 wild-type PDZ2 peptide along with their respective controls. A subset of these mice also received 4 mg/kg of the nitric oxide donor molsidomine. Hippocampal spine density, long-term potentiation, novel object recognition memory, and fear learning and memory were evaluated in mice. RESULTS: Exposure of 7-day-old mice to isoflurane or postsynaptic density-95 wild-type PDZ2 peptide relative to controls causes: (1) a long-term decrease in mushroom spines at 7 weeks (mean ± SD [spines per micrometer]): control (0.8 ± 0.2) versus isoflurane (0.4 ± 0.2), P < 0.0001, and PDZ2MUT (0.7 ± 0.2) versus PDZ2WT (0.4 ± 0.2), P < 0.001; (2) deficits in object recognition at 6 weeks (mean ± SD [recognition index]): naïve (70 ± 8) versus isoflurane (55 ± 14), P = 0.010, and control (65 ± 13) versus isoflurane (55 ± 14), P = 0.045, and PDZ2MUT (64 ±11) versus PDZ2WT (53 ± 18), P = 0.045; and (3) deficits in fear learning at 7 weeks and memory at 8 weeks (mean ± SD [% freezing duration]): Learning, control (69 ± 12) versus isoflurane (52 ± 13), P < 0.0001, and PDZ2MUT (65 ± 14) versus PDZ2WT (55 ± 14) P = 0.011, and Memory, control (80 ± 17) versus isoflurane (56 ± 23), P < 0.0001 and PDZ2MUT (73 ± 18) versus PDZ2WT (44 ± 19) P < 0.0001. Impairment in long-term potentiation has fully recovered here at 7 weeks (mean ± SD [% baseline]): control (140 ± 3) versus isoflurane (137 ± 8), P = 0.560, and PDZ2MUT (136 ± 17) versus PDZ2WT (128 ± 11), P = 0.512. The isoflurane induced decrease in mushroom spines was preventable by introduction of a nitric oxide donor. CONCLUSIONS: Early disruption of PDZ2 domain-mediated protein-protein interactions mimics isoflurane in decreasing mushroom spine density and causing learning and memory deficits in mice. Prevention of the decrease in mushroom spine density with a nitric oxide donor supports a role for neuronal nitric oxide synthase pathway in mediating this cellular change associated with cognitive impairment.

Stem cell-derived neurons from autistic individuals with SHANK3 mutation show morphogenetic abnormalities during early development.

Shank3 is a structural protein found predominantly at the postsynaptic density. Mutations in the SHANK3 gene have been associated with risk for autism spectrum disorder (ASD). We generated induced pluripotent stem cells (iPSCs) from control individuals and from human donors with ASD carrying microdeletions of SHANK3. In addition, we used Zinc finger nucleases to generate isogenic SHANK3 knockout human embryonic stem (ES) cell lines. We differentiated pluripotent cells into either cortical or olfactory placodal neurons. We show that patient-derived placodal neurons make fewer synapses than control cells. Moreover, patient-derived cells display a developmental phenotype: young postmitotic neurons have smaller cell bodies, more extensively branched neurites, and reduced motility compared with controls. These phenotypes were mimicked by SHANK3-edited ES cells and rescued by transduction with a Shank3 expression construct. This developmental phenotype is not observed in the same iPSC lines differentiated into cortical neurons. Therefore, we suggest that SHANK3 has a critical role in neuronal morphogenesis in placodal neurons and that early defects are associated with ASD-associated mutations.

The Protein Biochemistry of the Postsynaptic Density in Glutamatergic Synapses Mediates Learning in Neural Networks.

The strength of each excitatory synapse in the central nervous system is regulated by its prior activity in a process called synaptic plasticity. The initiation of synaptic plasticity occurs when calcium ions enter the postsynaptic compartment and encounter a subcellular structure called the postsynaptic density (PSD). The PSD is attached to the postsynaptic membrane just underneath the concentrated plaque of neurotransmitter receptors. It is comprised of a core set of 30-60 proteins, approximately 20 of which are scaffold proteins. The rest include protein kinases and phosphatases, some of which respond to calcium ion; small GTPases and their regulators; chaperones; ubiquitins; and proteases. The assembly of the PSD involves competitive binding among a variety of specific protein binding sites to form a dynamic network. A biochemical challenge for the future is to understand how the dynamic regulation of the structure, composition, and activity of the PSD mediates synaptic plasticity and how mutations in PSD proteins lead to mental and neurodegenerative diseases.

Inhibition of IL-6 trans-signaling in the brain increases sociability in the BTBR mouse model of autism.

Autism is a severe neurodevelopmental disorder with a large population prevalence, characterized by abnormal reciprocal social interactions, communication deficits, and repetitive behaviors with restricted interests. The BTBR T(+)Itpr3(tf) (BTBR) mice have emerged as strong candidates to serve as models of a range of autism-relevant behaviors. Increasing evidences suggest that interleukin (IL)-6, one of the most important neuroimmune factors, was involved in the pathophysiology of autism. It is of great importance to further investigate whether therapeutic interventions in autism can be achieved through the manipulation of IL-6. Our previous studies showed that IL-6 elevation in the brain could mediate autistic-like behaviors, possibly through the imbalances of neural circuitry and impairments of synaptic plasticity. In this study, we evaluate whether inhibiting IL-6 signaling in the brain is sufficient to modulate the autism-like behaviors on the BTBR mice. The results showed that chronic infusion of an analog of the endogenous IL-6 trans-signaling blocker sgp130Fc protein increased the sociability in BTBR mice. Furthermore, no change was observed in the number of excitatory synapse, level of synaptic proteins, density of dentitic spine and postsynaptic density in BTBR cortices after inhibiting IL-6 trans-signaling. However, inhibition of IL-6 trans-signaling increased the evoked glutamate release in synaptoneurosomes from the cerebral cortex of BTBR mice. Our findings suggest that inhibition of excessive production of IL-6 may have selective therapeutic efficacy in treating abnormal social behaviors in autism.

CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility.

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function.

Rabphilin 3A: A novel target for the treatment of levodopa-induced dyskinesias.

N-methyl-d-aspartate receptor (NMDAR) subunit composition strictly commands receptor function and pharmacological responses. Changes in NMDAR subunit composition have been documented in brain disorders such as Parkinson's disease (PD) and levodopa (L-DOPA)-induced dyskinesias (LIDs), where an increase of NMDAR GluN2A/GluN2B subunit ratio at striatal synapses has been observed. A therapeutic approach aimed at rebalancing NMDAR synaptic composition represents a valuable strategy for PD and LIDs. To this, the comprehension of the molecular mechanisms regulating the synaptic localization of different NMDAR subtypes is required. We have recently demonstrated that Rabphilin 3A (Rph3A) is a new binding partner of NMDARs containing the GluN2A subunit and that it plays a crucial function in the synaptic stabilization of these receptors. Considering that protein-protein interactions govern the synaptic retention of NMDARs, the purpose of this work was to analyse the role of Rph3A and Rph3A/NMDAR complex in PD and LIDs, and to modulate Rph3A/GluN2A interaction to counteract the aberrant motor behaviour associated to chronic L-DOPA administration. Thus, an array of biochemical, immunohistochemical and pharmacological tools together with electron microscopy were applied in this study. Here we found that Rph3A is localized at the striatal postsynaptic density where it interacts with GluN2A. Notably, Rph3A expression at the synapse and its interaction with GluN2A-containing NMDARs were increased in parkinsonian rats displaying a dyskinetic profile. Acute treatment of dyskinetic animals with a cell-permeable peptide able to interfere with Rph3A/GluN2A binding significantly reduced their abnormal motor behaviour. Altogether, our findings indicate that Rph3A activity is linked to the aberrant synaptic localization of GluN2A-expressing NMDARs characterizing LIDs. Thus, we suggest that Rph3A/GluN2A complex could represent an innovative therapeutic target for those pathological conditions where NMDAR composition is significantly altered.

Chronic stress-induced dendritic reorganization and abundance of synaptosomal PKA-dependent CP-AMPA receptor in the basolateral amygdala in a mouse model of depression.

Chronic stress is a precipitating factor for disorders including depression. The basolateral amygdala (BLA) is a critical substrate that interconnects with stress-modulated neural networks to generate emotion- and mood-related behaviors. The current study shows that 3 h per day of restraint stress for 14 days caused mice to exhibit long-term depressive behaviors, manifested by disrupted sociality and despair levels, which were rescued by fluoxetine. These behavioral changes corresponded with morphological and molecular changes in BLA neurons, including chronic stress-elicited increases in arborization, dendritic length, and spine density of BLA principal neurons. At the molecular level, calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (CP-AMPARs) within the synaptosome exhibited an increased GluR1:GluR2 subunit ratio. We also observed increased GluR1 phosphorylation at Ser 845 and enhanced cyclic AMP-dependent protein kinase (PKA) activity in the BLA. These molecular changes reverted to the basal state post-treatment with fluoxetine. The expression of synaptophysin (SYP) and postsynaptic density protein 95 (PSD-95) at BLA neuronal synapses was also enhanced by chronic stress, which was reversed post-treatment. Finally, chronic stress-provoked depressive behavior was overcome by local blockage of CP-AMPARs in the BLA via stereotaxic injection (IEM-1460). Chronic stress-elicited depressive behavior may be due to hypertrophy of BLA neuronal dendrites and increased of PKA-dependent CP-AMPAR levels in BLA neurons. Furthermore, fluoxetine can reverse chronic stress-triggered cytoarchitectural and functional changes of BLA neurons. These findings provide insights into depression-linked structural and functional changes in BLA neurons.

Proteomic Analysis of Mouse Cortex Postsynaptic Density following Neonatal Brain Hypoxia-Ischemia.

Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding of protein composition and signal networks in the adult brain, which underlie neuronal plasticity and neurodegenerative or psychiatric disorders. However, there is a paucity of knowledge about the architecture and organization of PSDs in the immature brain, and how it is modified by brain injury in an early developing stage. Mass spectrometry (MS)-based proteomic analysis was performed on PSDs prepared from cortices of postnatal day 9 naïve mice or pups which had suffered hypoxic-ischemic (HI) brain injury. 512 proteins of different functional groups were identified from PSDs collected 1 h after HI injury, among which 60 have not been reported previously. Seven newly identified proteins involved in neural development were highlighted. HI injury increased the yield of PSDs at early time points upon reperfusion, and multiple proteins were recruited into PSDs following the insult. Quantitative analysis was performed using spectral counting, and proteins whose relative expression was more than 50% up- or downregulated compared to the sham animals 1 h after HI insult were reported. Validation with Western blotting demonstrated changes in expression and phosphorylation of the N-methyl-D-aspartate receptor, activation of a series of postsynaptic protein kinases and dysregulation of scaffold and adaptor proteins in response to neonatal HI insult. This work, along with other recent studies of synaptic protein profiling in the immature brain, builds a foundation for future investigation on the molecular mechanisms underlying developing plasticity. Furthermore, it provides insights into the biochemical changes of PSDs following early brain hypoxia-ischemia, which is helpful for understanding not only the injury mechanisms, but also the process of repair or replenishment of neuronal circuits during recovery from brain damage.

Pathological Role of Peptidyl-Prolyl Isomerase Pin1 in the Disruption of Synaptic Plasticity in Alzheimer's Disease.

Synaptic loss is the structural basis for memory impairment in Alzheimer's disease (AD). While the underlying pathological mechanism remains elusive, it is known that misfolded proteins accumulate as ß-amyloid (Aß) plaques and hyperphosphorylated Tau tangles decades before the onset of clinical disease. The loss of Pin1 facilitates the formation of these misfolded proteins in AD. Pin1 protein controls cell-cycle progression and determines the fate of proteins by the ubiquitin proteasome system. The activity of the ubiquitin proteasome system directly affects the functional and structural plasticity of the synapse. We localized Pin1 to dendritic rafts and postsynaptic density (PSD) and found the pathological loss of Pin1 within the synapses of AD brain cortical tissues. The loss of Pin1 activity may alter the ubiquitin-regulated modification of PSD proteins and decrease levels of Shank protein, resulting in aberrant synaptic structure. The loss of Pin1 activity, induced by oxidative stress, may also render neurons more susceptible to the toxicity of oligomers of Aß and to excitation, thereby inhibiting NMDA receptor-mediated synaptic plasticity and exacerbating NMDA receptor-mediated synaptic degeneration. These results suggest that loss of Pin1 activity could lead to the loss of synaptic plasticity in the development of AD.

Proteomic and genomic evidence implicates the postsynaptic density in schizophrenia.

The postsynaptic density (PSD) contains a complex set of proteins of known relevance to neuropsychiatric disorders, and schizophrenia specifically. We enriched for this anatomical structure, in the anterior cingulate cortex, of 20 schizophrenia samples and 20 controls from the Stanley Medical Research Institute, and used unbiased shotgun proteomics incorporating label-free quantitation to identify differentially expressed proteins. Quantitative investigation of the PSD revealed more than 700 protein identifications and 143 differentially expressed proteins. Prominent among these were altered expression of proteins involved in clathrin-mediated endocytosis (CME) (Dynamin-1, adaptor protein 2) and N-methyl-D-aspartate (NMDA)-interacting proteins such as CYFIP2, SYNPO, SHANK3, ESYT and MAPK3 (all P<0.0015). Pathway analysis of the differentially expressed proteins implicated the cellular processes of endocytosis, long-term potentiation and calcium signaling. Both single-gene and gene-set enrichment analyses in genome-wide association data from the largest schizophrenia sample to date of 13,689 cases and 18,226 controls show significant association of HIST1H1E and MAPK3, and enrichment of our PSD proteome. Taken together, our data provide robust evidence implicating PSD-associated proteins and genes in schizophrenia, and suggest that within the PSD, NMDA-interacting and endocytosis-related proteins contribute to disease pathophysiology.

Melatonin attenuates impairments of structural hippocampal neuroplasticity in OXYS rats during active progression of Alzheimer's disease-like pathology.

Translational research on Alzheimer's disease (AD) has often focused on reducing the high cerebral levels of amyloid-ß (Aß) as a key characteristic of AD pathogenesis. There is, however, a growing body of evidence that synaptic dysfunction may be crucial for the development of the most common (sporadic) form of AD. The applicability of melatonin (mainly produced by the pineal gland) to the treatment of AD is actively evaluated, but usually, such studies are based on animal models of early-onset AD, which is responsible for only ~5% of AD cases. We have shown previously that in OXYS rats (an established model of sporadic AD), accumulation of toxic forms of Aß in the brain occurs later than does the development of signs of neurodegenerative changes and synaptic failure. In this regard, recently, we uncovered beneficial neuroprotective effects of melatonin (prophylactic dietary supplementation) in OXYS rats. Our aim here was to evaluate, starting at the age of active progression of AD-like pathology in OXYS rats, the effects of long-term oral administration of melatonin on the structure of synapses and on neuronal and glial cells of the hippocampus. Melatonin significantly increased hippocampal synaptic density and the number of excitatory synapses, decreased the number of inhibitory synapses, and upregulated pre- and postsynaptic proteins (synapsin I and PSD-95, respectively). Furthermore, melatonin improved the ultrastructure of neuronal and glial cells and reduced glial density. Based on our past and present results, the repair of neuroplasticity by melatonin is a promising strategy against AD.

Milnacipran remediates impulsive deficits in rats with lesions of the ventromedial prefrontal cortex.

BACKGROUND: Deficits in impulse control are often observed in psychiatric disorders in which abnormalities of the prefrontal cortex are observed, including attention-deficit/hyperactivity disorder and bipolar disorder. We recently found that milnacipran, a serotonin/noradrenaline reuptake inhibitor, could suppress impulsive action in normal rats. However, whether milnacipran could suppress elevated impulsive action in rats with lesions of the ventromedial prefrontal cortex, which is functionally comparable with the human prefrontal cortex, remains unknown. METHODS: Selective lesions of the ventromedial prefrontal cortex were made using quinolinic acid in rats previously trained on a 3-choice serial reaction time task. Sham rats received phosphate buffered saline. Following a period of recovery, milnacipran (0 or 10mg/kg/d × 14 days) was orally administered 60 minutes prior to testing on the 3-choice task. After 7 days of drug cessation, Western blotting, immunohistochemistry, electrophysiological analysis, and morphological analysis were conducted. RESULTS: Lesions of the ventromedial prefrontal cortex induced impulsive deficits, and repeated milnacipran ameliorated the impulsive deficit both during the dosing period and after the cessation of the drug. Repeated milnacipran remediated the protein levels of mature brain-derived neurotrophic factor and postsynaptic density-95, dendritic spine density, and excitatory currents in the few surviving neurons in the ventromedial prefrontal cortex of ventromedial prefrontal cortex-lesioned rats. CONCLUSIONS: The findings of this study suggest that milnacipran treatment could be a novel strategy for the treatment of psychiatric disorders that are associated with a lack of impulse control.

Disruption of the non-canonical Wnt gene PRICKLE2 leads to autism-like behaviors with evidence for hippocampal synaptic dysfunction.

Autism spectrum disorders (ASDs) have been suggested to arise from abnormalities in the canonical and non-canonical Wnt signaling pathways. However, a direct connection between a human variant in a Wnt pathway gene and ASD-relevant brain pathology has not been established. Prickle2 (Pk2) is a post-synaptic non-canonical Wnt signaling protein shown to interact with post-synaptic density 95 (PSD-95). Here, we show that mice with disruption in Prickle2 display behavioral abnormalities including altered social interaction, learning abnormalities and behavioral inflexibility. Prickle2 disruption in mouse hippocampal neurons led to reductions in dendrite branching, synapse number and PSD size. Consistent with these findings, Prickle2 null neurons show decreased frequency and size of spontaneous miniature synaptic currents. These behavioral and physiological abnormalities in Prickle2 disrupted mice are consistent with ASD-like phenotypes present in other mouse models of ASDs. In 384 individuals with autism, we identified two with distinct, heterozygous, rare, non-synonymous PRICKLE2 variants (p.E8Q and p.V153I) that were shared by their affected siblings and inherited paternally. Unlike wild-type PRICKLE2, the PRICKLE2 variants found in ASD patients exhibit deficits in morphological and electrophysiological assays. These data suggest that these PRICKLE2 variants cause a critical loss of PRICKLE2 function. The data presented here provide new insight into the biological roles of Prickle2, its behavioral importance, and suggest disruptions in non-canonical Wnt genes such as PRICKLE2 may contribute to synaptic abnormalities underlying ASDs.

Integrin ß1 signals through Arg to regulate postnatal dendritic arborization, synapse density, and behavior.

Integrins are heterodimeric extracellular matrix receptors that are essential for the proper development of the vertebrate nervous system. We report here that selective loss of integrin ß1 in excitatory neurons leads to reductions in the size and complexity of hippocampal dendritic arbors, hippocampal synapse loss, impaired hippocampus-dependent learning, and exaggerated psychomotor sensitivity to cocaine in mice. Our biochemical and genetic experiments demonstrate that the intracellular tail of integrin ß1 binds directly to Arg kinase and that this interaction stimulates activity of the Arg substrate p190RhoGAP, an inactivator of the RhoA GTPase. Moreover, genetic manipulations that reduce integrin ß1 signaling through Arg recapitulate the integrin ß1 knock-out phenotype in a gene dose-sensitive manner. Together, these results describe a novel integrin ß1-Arg-p190RhoGAP pathway that regulates dendritic arbor size, promotes synapse maintenance, supports proper hippocampal function, and mitigates the behavioral consequences of cocaine exposure.

De novo CNV analysis implicates specific abnormalities of postsynaptic signalling complexes in the pathogenesis of schizophrenia.

A small number of rare, recurrent genomic copy number variants (CNVs) are known to substantially increase susceptibility to schizophrenia. As a consequence of the low fecundity in people with schizophrenia and other neurodevelopmental phenotypes to which these CNVs contribute, CNVs with large effects on risk are likely to be rapidly removed from the population by natural selection. Accordingly, such CNVs must frequently occur as recurrent de novo mutations. In a sample of 662 schizophrenia proband-parent trios, we found that rare de novo CNV mutations were significantly more frequent in cases (5.1% all cases, 5.5% family history negative) compared with 2.2% among 2623 controls, confirming the involvement of de novo CNVs in the pathogenesis of schizophrenia. Eight de novo CNVs occurred at four known schizophrenia loci (3q29, 15q11.2, 15q13.3 and 16p11.2). De novo CNVs of known pathogenic significance in other genomic disorders were also observed, including deletion at the TAR (thrombocytopenia absent radius) region on 1q21.1 and duplication at the WBS (Williams-Beuren syndrome) region at 7q11.23. Multiple de novos spanned genes encoding members of the DLG (discs large) family of membrane-associated guanylate kinases (MAGUKs) that are components of the postsynaptic density (PSD). Two de novos also affected EHMT1, a histone methyl transferase known to directly regulate DLG family members. Using a systems biology approach and merging novel CNV and proteomics data sets, systematic analysis of synaptic protein complexes showed that, compared with control CNVs, case de novos were significantly enriched for the PSD proteome (P=1.72 × 10⁻6. This was largely explained by enrichment for members of the N-methyl-D-aspartate receptor (NMDAR) (P=4.24 × 10⁻6) and neuronal activity-regulated cytoskeleton-associated protein (ARC) (P=3.78 × 10⁻8) postsynaptic signalling complexes. In an analysis of 18 492 subjects (7907 cases and 10 585 controls), case CNVs were enriched for members of the NMDAR complex (P=0.0015) but not ARC (P=0.14). Our data indicate that defects in NMDAR postsynaptic signalling and, possibly, ARC complexes, which are known to be important in synaptic plasticity and cognition, play a significant role in the pathogenesis of schizophrenia.

Localization of fused in sarcoma (FUS) protein to the post-synaptic density in the brain.

Mutations in the fused in sarcoma (FUS) gene are linked to a form of familial amyotrophic lateral sclerosis (ALS), ALS6. The FUS protein is a major component of the ubiquitin-positive neuronal cytoplasmic inclusions in both ALS6 and some rare forms of frontotemporal lobar degeneration (FTLD). The latter are now collectively referred to as FTLD-FUS. In the present study, we investigated the localization of FUS in human and mouse brains. FUS was detected by western blot as an approximately 72 kDa protein in both human and mouse brains. Immunohistochemistry using lightly fixed tissue sections of human and mouse brains revealed FUS-positive granular staining in the neuropil, in addition to nuclear staining. Such granules are abundant in the gray matter of the brainstem and spinal cord. They are not frequent in the telencephalon. At the light microscopic level, FUS-positive granules are often co-localized with synaptophysin and present in association with microtubule-associated protein 2-positive dendrites. In the synaptosomal fraction of mouse brain, FUS is detected mainly in the post-synaptic density fraction. Thus, while FUS is primarily a nuclear protein, it may also play a role in dendrites. In the brains of patients with FTLD with TDP-43 deposition (FTLD-TDP), the number of FUS-positive granules in the cortex is increased compared with control cases. The increase in Alzheimer's disease (AD) is less remarkable but still significant. The dendritic localization of FUS and its increase in FTLD-TDP and AD may have some implication for the pathophysiology of neurodegenerative diseases.

NCAM/spectrin complex disassembly results in PSD perforation and postsynaptic endocytic zone formation.

Mechanisms inducing perforation of the postsynaptic density (PSD) are poorly understood. We show that neural cell adhesion molecule- deficient (NCAM-/-) hippocampal neurons have an abnormally high percentage of synapses with perforated PSDs. The percentage of synapses with perforated PSDs is also increased in wild-type (NCAM+/+) neurons after the disruption of the NCAM/spectrin complex indicating that the NCAM-assembled spectrin cytoskeleton maintains the structural integrity of PSDs. We demonstrate that PSD perforations contain endocytic zones involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) internalization. Induction of long-term potentiation in NCAM+/+ neurons accompanied by insertion of AMPAR into the neuronal cell surface is subsequently followed by formation of perforated synapses and AMPAR endocytosis suggesting that perforation of PSDs is important for membrane homeostasis in activated synapses. In NCAM-/- or NCAM+/+ neurons with dissociated spectrin meshwork, AMPAR endocytosis is enhanced under conditions of basal activity. An abnormally high rate of postsynaptic membrane endocytosis may thus contribute to brain pathologies associated with mutations in NCAM or spectrin.

Early changes in the synapses of the neostriatum induced by perinatal asphyxia.

Perinatal asphyxia (PA) is a medical condition associated with a high short-term morbimortality and different long-term neurological diseases. In previous work we have observed at 6 months post-synaptic densities (PSDs) alterations compatible with neurodegeneration highly correlated with the increment in the ubiquitination. Although alterations in the synaptic organization and function have been related with neuronal death after hypoxia, little is known about the synaptic changes in young animals exposed to PA. The main aim of this work is to study the PSDs changes in striatum of 30-day-old rats subjected to PA. Using two-dimensional electron microscopic analyses of synapses staining with ethanolic phosphotungstic acid we observed an increment of PSD thickness in severe hypoxic rats. These data are consistent with the western blot analysis that showed an increment in ubiquitination levels in the synapses of severe hypoxic rat. We did observe any alterations neither in synaptic structure nor in ubiquitinization in mild asphyctic rats. These data suggest that hypoxia might cause early misfolding and aggregation of synaptic proteins in severe anoxic animas that could induce long-term neurodegeneration.

NADPH oxidase-mediated oxidative damage to proteins in the postsynaptic density after transient cerebral ischemia and reperfusion.

NADPH oxidase is an important source of superoxide in the central nervous system. Although NADPH oxidase is localized near the postsynaptic site in neurons, little is known about the pathophysiological role of NADPH oxidase in synapses after cerebral ischemia and reperfusion. In the present study, we sought to determine the role of NADPH oxidase in oxidative damage to postsynaptic density (PSD) proteins, which were isolated from rats subjected to transient focal cerebral ischemia and reperfusion. The amounts of carbonylated PSD proteins were increased after transient focal cerebral ischemia and reperfusion. This change was accompanied by an increase in the level of NADPH oxidase subunits in the PSD. The administration of apocynin, an NADPH oxidase inhibitor, attenuated both the protein carbonylation in the PSD and cerebral infarct volume. We further demonstrated that the decreases seen in the amounts of PSD-associated proteins, such as neuroligin, N-cadherin, and SAP102, in the PSD were prevented by treatment with apocynin. These results suggest that pronounced activation of NADPH oxidase in the PSD after cerebral ischemia and reperfusion may be related to the focal oxidative damage to synaptic functions and subsequent development of ischemia and reperfusion-induced cerebral injury.