Membrane-associated Guanylate Kinase Family Research Articles

Neurodevelopmental disorders and the role of PSD-95: Understanding pathways and pharmacological interventions

Neurodevelopmental disorders (NDDs) are often linked to disruption in brain development and present challenges for affected individuals in achieving their cognitive, emotional, and motor developmental milestones. NDDs encompass a spectrum of conditions, including autism spectrum disorder (ASD), schizophrenia (SCZ), attention-deficit hyperactivity disorder (ADHD), and epilepsy. The unequivocal diagnosis of an NDD is often challenging due to overlapping signs and symptoms across different conditions. Synaptic plasticity, the activity-driven modification of synaptic strength and efficacy, plays a crucial role in brain network formation and organization and is frequently altered in NDDs. Here, we explore the multifaceted roles of postsynaptic density-95 kDa (PSD-95) in NDDs. Psd-95 is a scaffolding protein belonging to the membrane-associated guanylate kinases (MAGUKs) family, located at the core of synapses, and is central to synaptic plasticity. Dysregulation of PSD-95 is linked to various neuropsychiatric disorders. In SCZ, decreased PSD-95 expression affects synaptic plasticity, thereby impacting learning and memory. Genes associated with ASD interact with PSD-95, and its removal in mice leads to ASD-like behavioral abnormalities. Furthermore, PSD-95 is implicated in ADHD, where its modulation influences neurotransmission. Medications used in NDD treatment, such as antipsychotic drugs and selective serotonin reuptake inhibitors (SSRIs), can alter PSD-95 levels, potentially influencing synapse formation. Alpha-2 adrenergic agonists might enhance synaptic integrity by impacting PSD-95. Alternative pharmacotherapies such as memantine, allopurinol, and ketamine, all influencing PSD-95 to a certain extent, hold promise in managing NDDs. Understanding the role of PSD-95 in these disorders can deepen our biological comprehension and pave the way for targeted therapies. Specifically, exploring how PSD-95 affects synaptic plasticity and dendritic spine development could uncover opportunities for repurposing drugs to treat NDDs associated with mutations in the DLG4 gene encoding PSD-95.

ADAM22 ethnic-specific variant reducing binding of membrane-associated guanylate kinases causes focal epilepsy and behavioural disorder.

Pathogenic variants of ADAM22 affecting either its biosynthesis and/or its interactions with either LGI1 and/or PSD-95 have been recently identified in individuals with developmental and epileptic encephalopathy. Here, we describe a girl with seizures, delayed psychomotor development, and behavioural disorder, carrying a homozygous variant in ADAM22 (NM_021723.5:c.2714C > T). The variant has a surprisingly high frequency in the Roma population of the Czech and Slovak Republic, with 11 of 213 (∼5.2%) healthy Roma individuals identified as heterozygous carriers. Structural in silico characterization revealed that the genetic variant encodes the missense variant p.S905F, which localizes to the PDZ-binding motif of ADAM22. Studies in transiently transfected mammalian cells revealed that the variant has no effect on biosynthesis and stability of ADAM22. Rather, protein-protein interaction studies showed that the p.S905F variant specifically impairs ADAM22 binding to PSD-95 and other proteins from a family of membrane-associated guanylate kinases, while it has only minor effect on ADAM22-LGI1 interaction. Our study indicates that a significant proportion of epilepsy in patients of Roma ancestry may be caused by homozygous c.2714C > T variants in ADAM22. The study of this ADAM22 variant highlights a novel pathogenic mechanism of ADAM22 dysfunction and reconfirms an essential role of interaction of ADAM22 with membrane-associated guanylate kinases in seizure protection in humans.

Identification of membrane palmitoylated protein 1 (MPP1) as a heart-failure-promoting protein triggered by cardiovascular risk factors and aging

Membrane-Associated Guanylate Kinase (MAGUK) proteins are scaffold proteins with well-established functions in the neuronal system. A role of MAGUK protein up-regulation in the pathogenesis of heart failure is not established. This study identified the up-regulation of the MAGUK family protein MPP1 (Membrane Palmitoylated Protein 1), in cardiac transcriptome data of three different heart failure models. MPP1 was up-regulated in failing hearts of B6 mice with long-term chronic pressure overload, in failing hearts of aged Apoe−/− mice with long-term atherosclerosis, and in failing hearts of RKIP-transgenic mice with cardiotoxic lipid overload. MPP1-transgenic mice revealed that moderately (2-fold) increased cardiac MPP1 levels caused symptoms of heart failure with a significantly reduced left ventricular ejection fraction of 39.0 ± 6.9 % in Tg-MPP1 mice compared to 55.2 ± 3.7 % of non-transgenic B6 controls. Echocardiographic and histological analyses detected cardiac enlargement and cardiac dilation in Tg-MPP1 mice. The angiotensin II AT1 receptor (AGTR1) and MPP1 were co-localized on sarcolemmal membranes in vivo, and Tg-MPP1 mice had increased levels of cardiac AGTR1, which has an established heart failure-promoting function. The increased AGTR1 protein could be directly triggered by elevated MPP1 because MPP1 also increased the AGTR1 protein in non-cardiomyocyte HEK cells, which was detected by fluorescence measurement of AGTR1eYFP. MPP1 was not only up-regulated by major cardiovascular risk factors but also by old age, which is a major contributor to heart failure. Thus, the aging-induced MPP1 exerts a previously unrecognized role in heart failure pathogenesis by upregulation of the angiotensin II AT1 receptor (AGTR1) protein.

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal

The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

Recent Hints on the Dual Role of Discs Large MAGUK Scaffold Protein 5 in Cancers and in Hepatocellular Carcinoma.

Discs large MAGUK scaffold protein 5 (DLG5) is a multi-domain member of membrane-associated guanylate kinase (MAGUK) family, which plays a major role in the maintenance of cell epithelial polarity being part of the SCRIB-LGL-DLG complex. Although polarity proteins have been generally considered tumor suppressors, recent discoveries led to reconsidering their role in cancer. This is also true for DLG5 in different cancer types, including hepatocellular carcinoma (HCC). In this cancer, DLG5 was negatively associated with malignant characteristics, however recent findings associated DLG5 expression with advanced stages of HCC. In vitro studies evidenced its possible role in sustaining cell growth and migration by the interaction with several intracellular pathways, such as Hippo, Hedgehog, and PI3K/AKT signaling pathways. In this review, we summarize the recent finding on the dual role of DLG5 and other polarity proteins in cancers. What emerges is a still undefined role of those proteins in cancers, especially in HCC, one of the most frequent cancers worldwide, where the function of DLG5 and other polarity proteins is still largely unexplored.

Novel role of the synaptic scaffold protein Dlgap4 in ventricular surface integrity and neuronal migration during cortical development

Subcortical heterotopias are malformations associated with epilepsy and intellectual disability, characterized by the presence of ectopic neurons in the white matter. Mouse and human heterotopia mutations were identified in the microtubule-binding protein Echinoderm microtubule-associated protein-like 1, EML1. Further exploring pathological mechanisms, we identified a patient with an EML1-like phenotype and a novel genetic variation in DLGAP4. The protein belongs to a membrane-associated guanylate kinase family known to function in glutamate synapses. We showed that DLGAP4 is strongly expressed in the mouse ventricular zone (VZ) from early corticogenesis, and interacts with key VZ proteins including EML1. In utero electroporation of Dlgap4 knockdown (KD) and overexpression constructs revealed a ventricular surface phenotype including changes in progenitor cell dynamics, morphology, proliferation and neuronal migration defects. The Dlgap4 KD phenotype was rescued by wild-type but not mutant DLGAP4. Dlgap4 is required for the organization of radial glial cell adherens junction components and actin cytoskeleton dynamics at the apical domain, as well as during neuronal migration. Finally, Dlgap4 heterozygous knockout (KO) mice also show developmental defects in the dorsal telencephalon. We hence identify a synapse-related scaffold protein with pleiotropic functions, influencing the integrity of the developing cerebral cortex.

MO064: Expression Studies on Magi2 in Different FSGS Models

Abstract BACKGROUND AND AIMS MAGI2 is a tight junction protein and member of the membrane-associated guanylate kinase (MAGUK) family. In the glomerulus, it is exclusively expressed in podocytes and interacts with the slit diaphragm protein nephrin. Recent studies and podocyte-specific MAGI2 knockout mice have demonstrated the essential role of this protein in the glomerulus. The aim of this work was to study the expression of MAGI2 in different focal segmental glomerulosclerosis (FSGS) mouse models, in the GlomAssay, in a FSGS-like zebrafish model, and in human biopsies of patients suffering from minimal change disease and FSGS. Additionally, using CRISPR/Cas9 knockout of magi2 and nephrin in zebrafish, we investigated the influence of both proteins on each other’s localization. METHOD We used nephrotoxic serum (NTS) as well as uninephrectomy DOCA/salt-induced hypertension as murine models. The podocyte-specific nitroreductase/metronidazole zebrafish model was used to induce FSGS-like disease in zebrafish upon partial podocyte-depletion. MAGI2 expression was evaluated in kidney biopsies of patients affected by minimal change disease (MCD), primary and secondary FSGS. Slides were imaged using confocal laser scanning and super-resolution 3D-structured illumination microscopy (3D-SIM). Filtration slit density (FSD) was measured on human biopsies by PEMP (podocyte exact morphology measurement procedure). MAGI2/nephrin ratio measurements were performed with a customized macro for the ImageJ-based platform FIJI. Magi2 expression was studied in the GlomAssay. To evaluate the interaction between magi2 and nephrin in the zebrafish model, the respective proteins were knocked-out using CRISPR/Cas9. The knockout was validated by RT–PCR and the expression of these two proteins in both lines was analyzed by immunofluorescence staining. Proteinuria was assessed using the excretion of eGFP-tagged vitamin D-binding protein from the vasculature and reuptake in proximal tubule cells. RESULTS MAGI2 was expressed in a linear pattern along the filtration slit and colocalized with nephrin in all three species investigated (human, mouse and zebrafish) (3D-SIM in Fig. 1). MAGI2 was significantly downregulated in NTS-induced nephritis, as well as in uninephrectomized and DOCA/salt-treated mice. In the GlomAssay, Magi2 was significantly downregulated in dedifferentiated podocytes after 6 days in cell culture due to podocyte dedifferentiation. A reduction of MAGI2 was found in human biopsies of patients suffering from primary FSGS, but not in MCD and secondary FSGS, respectively. In addition, we found that magi2 was downregulated in zebrafish larvae using our FSGS-like zebrafish model. When we examined the effect of nephrin on magi2 expression and vice versa, we found that magi2 expression was unchanged in nephrin-KO larvae, whereas nephrin expression was significantly reduced in MAGI2a-KO larvae. CONCLUSION Here, we have shown that the expression of MAGI2 in the glomerulus of different species such as zebrafish, mice and humans is associated with the expression of nephrin. By using knockout larvae, we showed that nephrin expression is dependent on Magi2 and not vice versa. Since the expression of Magi2 seems to be specifically regulated depending on the podocytopathy investigated, we propose a role for MAGI2 to distinguish between different glomerulopathies.

Human Serine Racemase Weakly Binds the Third PDZ Domain of PSD-95.

Human serine racemase (hSR) is a pyridoxal-5′-phosphate (PLP)-dependent dimer that catalyzes the formation of D-serine from L-serine, as well as the dehydration of both L- and D-serine to pyruvate and ammonia. As D-serine is a co-agonist of N-methyl-D-aspartate receptors (NMDARs), hSR is a key enzyme in glutamatergic neurotransmission. hSR activity is finely regulated by Mg2+, ATP, post-translational modifications, and the interaction with protein partners. In particular, the C-terminus of murine SR binds the third PDZ domain (PDZ3) of postsynaptic density protein 95 (PSD-95), a member of the membrane-associated guanylate kinase (MAGUK) family involved in the trafficking and localization of glutamate receptors. The structural details of the interaction and the stability of the complex have not been elucidated yet. We evaluated the binding of recombinant human PSD-95 PDZ3 to hSR by glutaraldehyde cross-linking, pull-down assays, isothermal titration calorimetry, nuclear magnetic resonance, and enzymatic assays. Overall, a weak interaction was observed, confirming the binding for the human orthologs but supporting the hypothesis that a third protein partner (i.e., stargazin) is required for the regulation of hSR activity by PSD-95 and to stabilize their interaction.

Deletion of p38γ attenuates ethanol consumption- and acetaminophen-induced liver injury in mice through promoting Dlg1.

Acetaminophen (APAP) is one of the major causes of drug-induced acute liver injury, and ethanol may aggravate APAP-induced liver injury. The problem of ethanol- and APAP-induced liver injury becomes increasingly prominent, but the mechanism of ethanol- and APAP-induced liver injury remains ambiguous. p38γ is one of the four isoforms of P38 mitogen activated protein kinases, that contributes to inflammation in different diseases. In this study we investigated the role of p38γ in ethanol- and APAP-induced liver injury. Liver injury was induced in male C57BL/6 J mice by giving liquid diet containing 5% ethanol (v/v) for 10 days, followed by gavage of ethanol (25% (v/v), 6 g/kg) once or injecting APAP (200 mg/kg, ip), or combined the both treatments. We showed that ethanol significantly aggravated APAP-induced liver injury in C57BL/6 J mice. Moreover, the expression level of p38γ was up-regulated in the liver of ethanol-, APAP- and ethanol+APAP-treated mice. Knockdown of p38γ markedly attenuated liver injury, inflammation, and steatosis in ethanol+APAP-treated mice. Liver sections of p38γ-knockdown mice displayed lower levels of Oil Red O stained dots and small leaky shapes. AML-12 cells were exposed to APAP (5 mM), ethanol (100 mM) or combined treatments. We showed that P38γ was markedly increased in ethanol+APAP-treated AML-12 cells, whereas knockdown of p38γ significantly inhibited inflammation, lipid accumulation and oxidative stress in ethanol+APAP-treated AML-12 cells. Furthermore, we revealed that p38γ could combine with Dlg1, a member of membrane-associated guanylate kinase family. Deletion of p38γ up-regulated the expression level of Dlg1 in ethanol+APAP-treated AML-12 cells. In summary, our results suggest that p38γ functions as an important regulator in ethanol- and APAP-induced liver injury through modulation of Dlg1.

Design and Development of a Chemical Probe for Pseudokinase Ca2+/calmodulin-Dependent Ser/Thr Kinase.

CASK (Ca2+/calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.

Functional analysis of CASK transcript variants expressed in human brain.

The calcium-/calmodulin dependent serine protein kinase (CASK) belongs to the membrane-associated guanylate kinases (MAGUK) family of proteins. It fulfils several different cellular functions, ranging from acting as a scaffold protein to transcription control, as well as regulation of receptor sorting. CASK functions depend on the interaction with a variety of partners, for example neurexin, liprin-α, Tbr1 and SAP97. So far, it is uncertain how these seemingly unrelated interactions and resulting functions of CASK are regulated. Here, we show that alternative splicing of CASK can guide the binding affinity of CASK isoforms to distinct interaction partners. We report seven different variants of CASK expressed in the fetal human brain. Four out of these variants are not present in the NCBI GenBank database as known human variants. Functional analyses showed that alternative splicing affected the affinities of CASK variants for several of the tested interaction partners. Thus, we observed a clear correlation of the presence of one splice insert with poor binding of CASK to SAP97, supported by molecular modelling. The alternative splicing and distinct properties of CASK variants in terms of protein-protein interaction should be taken into consideration for future studies.

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Molecular Genetic Dissection of Inflammatory Linear Verrucous Epidermal Naevus Leads to Successful Targeted Therapy

Characterizing the Binding Sites for GK Domain of DLG1 and DLG4 via Molecular Dynamics Simulation.

Discs-large (DLG) is a member that belongs to the membrane-associated guanylate kinase (MAGUK) family. The GK domain of DLGs has evolved into a protein–protein interaction module that could bind with kinds of proteins to regulate diverse cellular functions. Previous reports have demonstrated the GK domain of DLGs functioned as a phosphor-peptide-binding module by resolving the crystal structures. Here we investigated into the interactions of DLG1 and DLG4 with their reported phosphor-peptides by molecular dynamics simulations. Post-dynamics analysis showed that DLG1/4 formed extensive interactions with phosphorylated ligands, including hydrophobic and hydrogen bonding interactions. Among them, the highly conserved residues among the DLGs in phosphor-site and β5 sheet were crucial for the binding according to the energy decomposition calculations. Additionally, the binding interactions between DLG4 and reported unphosphorylated peptides including MAP1A and designed GK inhibitory (GKI-QSF) peptides were analyzed. We found the key residues that played important roles in DLG4/unphosphorylated peptide systems were very similar as in DLG4/phosphor-peptide systems. Moreover, the molecular dynamic simulation for the complex of DLG1 and GKI-QSF was carried out and predicted that the GKI-QSF could bind with DLG1 with similar Kd value compared to DLG4/GKI-QSF, which was verified by using ITC assay (Kd = 1.20 ± 0.29 μM). Our study might be helpful for the better understanding of the structural and biological function of DLGs GK domain and encourage the discovery of new binders.

MAGI-2 downregulation: a potential predictor of tumor progression and early recurrence in Han Chinese patients with prostate cancer.

Membrane-associated guanylate kinase (MAGUK) family protein MAGUK invert 2 (MAGI-2) has been demonstrated to be involved in the tumorigenic mechanism of prostate cancer. The objective of this study was to investigate the expression of MAGI-2 at mRNA and protein levels. The prognostic value of MAGI-2 in Han Chinese patients with prostate cancer was also investigated. The expression data of MAGI-2 were assessed through database retrieval, analysis of sequencing data from our group, and tissue immunohistochemistry using digital scoring system (H-score). The clinical, pathological, and follow-up data were collected. The expression of MAGI-2 in prostate tumor tissues and prostate normal tissues was evaluated and compared. MAGI-2 expression was associated with clinical parameters including tumor stage, lymph node status, Gleason score, PSA level, and biochemical recurrence of prostate cancer. The relative expression of MAGI-2 mRNA was lower in the tumor tissue in The Cancer Genome Atlas (TCGA) database and sequencing data (P < 0.001). There was no difference in MAGI-2 protein expression between tumor and normal tissues in tissue microarray (TMA) results. MAGI-2 expression was associated with pathological tumor stage (P = 0.02), Gleason score (P = 0.05), and preoperation prostate-specific antigen (PSA; P = 0.04). A positive correlation was identified between MAGI-2 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expressions through the analysis of TCGA and TMA data (P < 0.0001). Patients with higher MAGI-2 expression had longer biochemical recurrence-free survival in the univariate analysis (P = 0.005), which indicates an optimal prognostic value of MAGI-2 in Han Chinese patients with prostate cancer. In conclusion, MAGI-2 expression gradually decreases with tumor progression, and can be used as a predictor of tumor recurrence in Chinese patients.

Slitrk2 controls excitatory synapse development via PDZ-mediated protein interactions

Members of the Slitrk (Slit- and Trk-like protein) family of synaptic cell-adhesion molecules control excitatory and inhibitory synapse development through isoform-dependent extracellular interactions with leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs). However, how Slitrks participate in activation of intracellular signaling pathways in postsynaptic neurons remains largely unknown. Here we report that, among the six members of the Slitrk family, only Slitrk2 directly interacts with the PDZ domain-containing excitatory scaffolds, PSD-95 and Shank3. The interaction of Slitrk2 with PDZ proteins is mediated by the cytoplasmic COOH-terminal PDZ domain-binding motif (Ile-Ser-Glu-Leu), which is not found in other Slitrks. Mapping analyses further revealed that a single PDZ domain of Shank3 is responsible for binding to Slitrk2. Slitrk2 forms in vivo complexes with membrane-associated guanylate kinase (MAGUK) family proteins in addition to PSD-95 and Shank3. Intriguingly, in addition to its role in synaptic targeting in cultured hippocampal neurons, the PDZ domain-binding motif of Slitrk2 is required for Slitrk2 promotion of excitatory synapse formation, transmission, and spine development in the CA1 hippocampal region. Collectively, our data suggest a new molecular mechanism for conferring isoform-specific regulatory actions of the Slitrk family in orchestrating intracellular signal transduction pathways in postsynaptic neurons.

Affinity proteomics identifies novel functional modules related to adhesion GPCRs.

Adhesion G protein-coupled receptors (ADGRs) have recently become a target of intense research. Their unique protein structure, which consists of a G protein-coupled receptor combined with long adhesive extracellular domains, suggests a dual role in cell signaling and adhesion. Despite considerable progress in the understanding of ADGR signaling over the past years, the knowledge about ADGR protein networks is still limited. For most receptors, only a few interaction partners are known thus far. We aimed to identify novel ADGR-interacting partners to shed light on cellular protein networks that rely on ADGR function. For this, we applied affinity proteomics, utilizing tandem affinity purifications combined with mass spectrometry. Analysis of the acquired proteomics data provides evidence that ADGRs not only have functional roles at synapses but also at intracellular membranes, namely at the endoplasmic reticulum, the Golgi apparatus, mitochondria, and mitochondria-associated membranes (MAMs). Specifically, we found an association of ADGRs with several scaffold proteins of the membrane-associated guanylate kinases family, elementary units of the γ-secretase complex, the outer/inner mitochondrial membrane, MAMs, and regulators of the Wnt signaling pathways. Furthermore, the nuclear localization of ADGR domains together with their physical interaction with nuclear proteins and several transcription factors suggests a role of ADGRs in gene regulation.

Discs large homolog 1 regulates B-cell proliferation and antibody production.

Antibody production results from B-cell activation and proliferation upon antigen binding. Discs large homolog 1 (Dlg1), a scaffold protein from the membrane-associated guanylate kinase family, has been shown to regulate the antigen receptor signaling and cell polarity in lymphocytes; however, the physiological function of Dlg1 in humoral responses is not completely clear. Here, we addressed this question using a conditional knockout (KO) mouse model with Dlg1 deficiency in different B-cell subsets by crossing dlg1fl/fl mice with either mb1cre/+ or aicdacre/+ mice, respectively. In both mouse models, we observed that Dlg1 deficiency in B cells (Dlg1-KO B cells) led to obvious hyper-antibody responses upon immunization, the effect of which was more obvious in antigen-recall responses. Mechanistically, we found that Dlg1-KO B cells exhibited hyper-proliferation compared with wild-type B cells upon antigen stimulation, suggesting that the hyper-antibody responses are likely induced by the hyper-proliferation of Dlg1-KO B cells. Indeed, further studies demonstrated that Dlg1 deficiency in B cells led to the down-regulation of a tumor suppressor, FoxO1. Thus, all these results reveal an unexpected function of Dlg1 in restraining hyper-antibody responses through the inhibition of FoxO1 and thus antigen-binding-induced proliferation in B cells.

Csmd2 Is a Synaptic Transmembrane Protein that Interacts with PSD-95 and Is Required for Neuronal Maturation.

Mutations and copy number variants of the CUB and Sushi multiple domains 2 (CSMD2) gene are associated with neuropsychiatric disease. CSMD2 encodes a single-pass transmembrane protein with a large extracellular domain comprising repeats of CUB and Sushi domains. High expression of CSMD2 in the developing and mature brain suggests possible roles in neuron development or function, but the cellular functions of CSMD2 are not known. In this study, we show that mouse Csmd2 is expressed in excitatory and inhibitory neurons in the forebrain. Csmd2 protein exhibits a somatodendritic localization in the neocortex and hippocampus, with smaller puncta localizing to the neuropil. Using immunohistochemical and biochemical methods, we demonstrate that Csmd2 localizes to dendritic spines and is enriched in the postsynaptic density (PSD). Accordingly, we show that the cytoplasmic tail domain of Csmd2 interacts with synaptic scaffolding proteins of the membrane-associated guanylate kinase (MAGUK) family. The association between Csmd2 and MAGUK member PSD-95 is dependent on a PDZ-binding domain on the Csmd2 tail, which is also required for synaptic targeting of Csmd2. Finally, we show that knock-down of Csmd2 expression in hippocampal neuron cultures results in reduced complexity of dendritic arbors and deficits in dendritic spine density. Knock-down of Csmd2 in immature developing neurons results in reduced filopodia density, whereas Csmd2 knock-down in mature neurons causes significant reductions in dendritic spine density and dendrite complexity. Together, these results point toward a function for Csmd2 in development and maintenance of dendrites and synapses, which may account for its association with certain psychiatric disorders.

Expression of CASK Antibody in Non-Mucinous Colorectal Adenocarcinoma

AbstractBackground: Colorectal cancers are one of the leading causes of cancer related morbidity and mortality worldwide. In Egypt was 4% in the year 2012 ranking as the sixth cancer in Egypt and representing 53% of GI malignancy. It is obvious that there is no single determining factor for the process of carcinogenesis or spread of colorectal carcinoma. There are many proposed contributing factors, including disturbances in Epithelial-Mesenchymal Transition (EMT) and loss of regulation of adhesion molecules and polarity proteins. Cal-cium/calmodulin-dependent serine protein kinase (CASK) belongs to the Membrane-Associated Guanylate Kinase (MAGUK) family. The role of CASK in Colorectal cancer hasn't been yet fully understood.Aim of Study: To evaluate the expression of Calci-um/calmodulin-dependent serine protein kinase (CASK) in non-mucinous colorectal adenocarcinoma and to evaluate its correlation with variable clinicopathological factors.Material and Methods: It is retrospective descriptive study is conducted in the Department of Pathology, Faculty of Medicine, Suez Canal University Teaching Hospital. It include 42 archived paraffin blocks of non-mucinous colorectal adenocarcinoma of patients who underwent surgical excision without previous chemotherapy or radiation, 15 adenomatous polyps and 5 normal mucosa samples. The clinical and path-ological data of studied patients are taken from medical records, pathology referral reports and pathology reports. Patients with incomplete data excluded from the study. Re-viewing the data of survival from the archived registry files at department of Oncology, Suez Canal University Teaching Hospital. Sections cut into 5um thick sections and mounted on positive charged slides for immunohistochemical staining using CASK Antibody (S56A-50): Mouse Monoclonal, Novus Biologicals, Catalogue no. NBP1-47648. Staining intensity graded according to the Allred score on a 0-3 scale.Data Analysis: The relation between CASK antibody staining intensity in the studied biopsies and other clinico-pathological parameters including (age, gender, histopatho-logical tumor features) was evaluated using Pearson's x2 test. The level of statistical significance (p-value) was set at Results: The study revealed that CASK protein had mainly cytoplasmic and cytoplasmic-membranous pattern of expres-sion. The CASK protein was overexpressed in the majority of CRC samples with 85.7% of cases showing moderate to strong expression, while only 14.3% of cases displayed minimal faint expression. On the other hand, CASK expression was low in the majority (53.3%) of adenoma samples. Addi-tionally, adenomas with high grade dysplasia showed stronger staining intensity for CASK protein and higher percentage of positive cases than the ones with low grade dysplasia by IHC. As regard to the association between CASK protein overex-pression and clinicopathological prognostic factors we found that CASK demonstrated significantly higher expression in tumor samples with early stages (I/II) rather than advanced stage (III/IV). Low grade tumors showed higher percentage of positive cases and stronger intensity of staining for CASK protein than high grade tumors. Expression of CASK protein showed no statistically significant correlation with patients' gender or age respectively. There was also no significant correlation with tumor site, gross tumor size, histologic tumor border configuration, lymphovascular invasion or distant metastasis.Conclusion: CASK protein was overexpressed in the majority of non-mucinous colorectal adenocarcinoma (CRC) cases with mainly cytoplasmic and cytoplasmic-membranous localization. However, its expression was significantly less in adenomas with a possible association with the grade of dysplasia; giving positive pattern in highly dysplastic cases suggesting a role in the progression of adenomas into carci-nomas. CASK overexpression also associated with both TNM stage of tumors and their histologic grade. CASK was signif-icantly overexpressed in early stage and low grade tumors rather than tumors with advanced stage and higher histological grades. This suggests that CASK protein is a good prognostic factor and might contribute in tumor confinement and local-ization.

An opposing function of paralogs in balancing developmental synapse maturation.

The disc-large (DLG)–membrane-associated guanylate kinase (MAGUK) family of proteins forms a central signaling hub of the glutamate receptor complex. Among this family, some proteins regulate developmental maturation of glutamatergic synapses, a process vulnerable to aberrations, which may lead to neurodevelopmental disorders. As is typical for paralogs, the DLG-MAGUK proteins postsynaptic density (PSD)-95 and PSD-93 share similar functional domains and were previously thought to regulate glutamatergic synapses similarly. Here, we show that they play opposing roles in glutamatergic synapse maturation. Specifically, PSD-95 promoted, whereas PSD-93 inhibited maturation of immature α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid–type glutamate receptor (AMPAR)–silent synapses in mouse cortex during development. Furthermore, through experience-dependent regulation of its protein levels, PSD-93 directly inhibited PSD-95’s promoting effect on silent synapse maturation in the visual cortex. The concerted function of these two paralogs governed the critical period of juvenile ocular dominance plasticity (jODP), and fine-tuned visual perception during development. In contrast to the silent synapse–based mechanism of adjusting visual perception, visual acuity improved by different mechanisms. Thus, by controlling the pace of silent synapse maturation, the opposing but properly balanced actions of PSD-93 and PSD-95 are essential for fine-tuning cortical networks for receptive field integration during developmental critical periods, and imply aberrations in either direction of this process as potential causes for neurodevelopmental disorders.