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SGEF (also known as ARHGEF26), a RhoG specific GEF, can form a ternary complex with the Scribble polarity complex proteins Scribble and Dlg1, which regulates the formation and maintenance of adherens junctions and barrier function of epithelial cells. Notably, silencing SGEF results in a dramatic downregulation of both E-cadherin and ZO-1 (also known as TJP1) protein levels. However, the molecular mechanisms involved in the regulation of this pathway are not known. Here, we describe a novel signaling pathway governed by the Scribble-SGEF-Dlg1 complex. Our results show that the three members of the ternary complex are required to maintain the stability of the apical junctions, ZO-1 protein levels and tight junction (TJ) permeability. In contrast, only SGEF is necessary to regulate E-cadherin levels. The absence of SGEF destabilizes the E-cadherin-catenin complex at the membrane, triggering a positive feedback loop that exacerbates the phenotype through the repression of E-cadherin transcription in a process that involves the internalization of E-cadherin by endocytosis, β-catenin signaling and the transcriptional repressor Slug (also known as SNAI2)., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Gallbladder cancer (GBC) is a highly malignant and rapidly progressing tumor of the human biliary system, and there is an urgent need to develop new therapeutic targets and modalities. Non-POU domain-containing octamer-binding protein (NONO) is an RNA-binding protein involved in the regulation of transcription, mRNA splicing, and DNA repair. NONO expression is elevated in multiple tumors and can act as an oncogene to promote tumor progression. Here, we found that NONO was highly expressed in GBC and promoted tumor cells growth. The dysregulation of RNA splicing is a molecular feature of almost all tumor types. Accordingly, mRNA-seq and RIP-seq analysis showed that NONO promoted exon6 skipping in DLG1, forming two isomers (DLG1-FL and DLG1-S). Furthermore, lower Percent-Spliced-In (PSI) values of DLG1 were detected in tumor tissue relative to the paraneoplastic tissue, and were associated with poor patient prognosis. Moreover, DLG1-S and DLG1-FL act as tumor promoters and tumor suppressors, respectively, by regulating the YAP1/JUN pathway. N6-methyladenosine (m6A) is the most common and abundant RNA modification involved in alternative splicing processes. We identified an m6A reader, IGF2BP3, which synergizes with NONO to promote exon6 skipping in DLG1 in an m6A-dependent manner. Furthermore, IP/MS results showed that RBM14 was bound to NONO and interfered with NONO-mediated exon6 skipping of DLG1. In addition, IGF2BP3 disrupted the binding of RBM14 to NONO. Overall, our data elucidate the molecular mechanism by which NONO promotes DLG1 exon skipping, providing a basis for new therapeutic targets in GBC treatment., Competing Interests: Declaration of competing interest There are no conflicts of interest to disclose. All authors have seen and approved the manuscript and consent publication., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Our previous study data suggested that the synapse-associated protein 97 (SAP97) rs3915512 polymorphism is significantly related to clinical performance in schizophrenia. The cerebellum exhibits abundant expression of SAP97, which is involved with negative symptoms, cognition and emotion in schizophrenia. As functional dysconnectivity with the cortical-subcortical-cerebellar circuitry has been widely shown in patients with schizophrenia, cortical-subcortical-cerebellar dysconnectivity can therefore be considered a possible intermediate phenotype that connects risk genes with schizophrenia. In this study, resting-state functional magnetic resonance imaging (fMRI) was applied to evaluate whether the SAP97 rs3915512 polymorphism changes cortical/subcortical-cerebellar resting-state functional connectivity (RSFC) in 104 Han Chinese subjects (52 first-episode schizophrenia (FES) patients and 52 matched healthy controls (HCs)). To examine RSFC between cortical/subcortical regions and the cerebellum, a ROI (region of interest)-wise functional connectivity analysis was conducted. The association between abnormal cortical/subcortical-cerebellar connectivity and clinical manifestation was further assessed in FES patients with different genotypes. The interactive effect of disease and genotype on RSFC was found between the frontal gyrus (rectus) and cerebellum. A positive correlation was suggested between RSFC in the cerebellum and the hostility scores in FES patients with the A allele, and no correlation was found in FES patients with the TT genotype. The current findings identified that SAP97 may be involved in the process of mental symptoms in FES patients via cerebellar connectivity depending on the rs3915512 polymorphism genotype., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Gap junction channels, composed of connexins, allow direct cell-to-cell communication. Connexin 43 (Cx43; also known as GJA1) is widely expressed in tissues, including the epidermis. In a previous study of human papillomavirus-positive cervical epithelial tumour cells, we identified Cx43 as a binding partner of the human homologue of Drosophila Discs large (Dlg1; also known as SAP97). Dlg1 is a member of the membrane associated-guanylate kinase (MAGUK) scaffolding protein family, which is known to control cell shape and polarity. Here, we show that Cx43 also interacts with Dlg1 in uninfected keratinocytes in vitro and in keratinocytes, dermal cells and adipocytes in normal human epidermis in vivo. Depletion of Dlg1 in keratinocytes did not alter Cx43 transcription but was associated with a reduction in Cx43 protein levels. Reduced Dlg1 levels in keratinocytes resulted in a reduction in Cx43 at the plasma membrane with a concomitant reduction in gap junctional intercellular communication and relocation of Cx43 to the Golgi compartment. Our data suggest a key role for Dlg1 in maintaining Cx43 at the plasma membrane in keratinocytes., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Background: Brugada syndrome (BrS) is an inherited primary channelopathy syndrome associated to sudden cardiac death. Overall, variants have been identified in eighteen genes encoding for ion channel subunits and seven genes for regulatory proteins. Recently, a missense variant in DLG1 has been found within a BrS phenotype-positive patient. DLG1 encodes for synapse associated protein 97 (SAP97), a protein characterized by the presence of multiple domains for protein-protein interactions including PDZ domains. In cardiomyocytes, SAP97 interacts with Nav1.5, a PDZ binding motif of SCN5A and others potassium channel subunits., Aim of the Study: To characterize the phenotype of an Italian family with BrS syndrome carrying a DLG1 variant., Methods: Clinical and genetic investigations were performed. Genetic testing was performed with whole-exome sequencing (WES) using the Illumina platform. According to the standard protocol, a variant found by WES was confirmed in all members of the family by bi-directional capillary Sanger resequencing. The effect of the variant was investigated by using in silico prediction of pathogenicity., Results: The index case was a 74-year-old man with spontaneous type 1 BrS ECG pattern that experienced syncope and underwent ICD implantation. WES of the index case, performed assuming a dominant mode of inheritance, identified a heterozygous variant, c.1556G>A (p.R519H), in the exon 15 of the DLG1 gene. In the pedigree investigation, 6 out of 12 family members had the variant. Carriers of the gene variant all had BrS ECG type 1 drug induced and showed heterogeneous cardiac phenotypes with two patients experiencing syncope during exercise and fever, respectively. The amino acid residue #519 lies near a PDZ domain and in silico analysis suggested a causal role for the variant. Modelling of the resulting protein structure predicted that the variant disrupts an H-bond and a likelihood of being pathogenic. As a consequence, it is likely that a conformational change affects protein functionality and the modulating role on ion channels., Conclusions: A DLG1 gene variant identified was associated with BrS. The variant could modify the formation of multichannel protein complexes, affecting ion channels to specific compartments in cardiomyocytes.

Despite advances in the treatment of multiple myeloma in the past decades, the disease remains incurable, and understanding signals and molecules that can control myeloma growth and survival are important for the development of novel therapeutic strategies. One such molecule, CD86, regulates multiple myeloma cell survival via its interaction with CD28 and signaling through its cytoplasmic tail. Although the CD86 cytoplasmic tail has been shown to be involved in drug resistance and can induce molecular changes in multiple myeloma cells, its function has been largely unexplored. Here, we show that CD86 cytoplasmic tail has a role in trafficking CD86 to the cell surface. This is due in part to a PDZ-binding motif at its C-terminus which is important for proper trafficking from the Golgi apparatus. BioID analysis revealed 10 PDZ domain-containing proteins proximal to CD86 cytoplasmic tail in myeloma cells. Among them, we found the planar cell polarity proteins, SCRIB and DLG1, are important for proper CD86 surface expression and the growth and survival of myeloma cells. These findings indicate a mechanism by which myeloma cells confer cellular survival and drug resistance and indicate a possible motif to target for therapeutic gain., Implications: These findings demonstrate the importance of proper trafficking of CD86 to the cell surface in myeloma cell survival and may provide a new therapeutic target in this disease., (©2022 American Association for Cancer Research.)

Aim: To investigate the long non-coding RNA DLG1 Antisense RNA 1 (lncRNA DLG1-AS1) mechanism in cervical cancer cells with gemcitabine (GEM) resistance., Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect DLG1-AS1, miR-16-5p, and hepatoma-derived growth factor (HDGF) expression in cervical cancer cells. The effects of DLG1-AS1 knockdown on cell viability, proliferation, and apoptosis were investigated in GEM-resistant cervical cancer cells. The binding of DLG1-AS1 with miR-16-5p and of miR-16-5p with HDGF was confirmed through dual-luciferase reporter assays. HDGF expression was detected through Western blotting. A xenograft model was established using stably transfected GEM-resistant cervical cancer cells to detect the role of DLG1-AS1 in tumorigenesis in vivo., Results: DLG1-AS1 expression was significantly elevated in HeLa/GEM and SiHa/GEM cells. DLG1-AS1 silencing significantly reduced the viability and proliferation of GEM-resistant cervical cancer cells. DLG1-AS1 also promoted GEM sensitivity in cervical cancer cells by inhibiting miR-16-5p. Moreover, the tumor volume in nude mice in the DLG1-AS1 knockdown group decreased after GEM treatment. In addition, DLG1-AS1 targeted miR-16-5p, and miR-16-5p targeted HDGF. The miR-16-5p inhibitor reversed the DLG1-AS1 knockdown effect in GEM-resistant cervical cancer cells., Conclusion: Knockdown of DLG1-AS1 promoted GEM sensitivity in cervical cancer cells by regulating miR-16-5p/HDGF., (© 2022 Japan Society of Obstetrics and Gynecology.)

Schizophrenia (SCZ) is a mental illness characterized by aberrant synaptic plasticity and connectivity. A large bulk of evidence suggests genetic and functional links between postsynaptic abnormalities and SCZ. Here, we performed quantitative PCR and Western blotting analysis in the dorsolateral prefrontal cortex (DLPFC) and hippocampus of SCZ patients to investigate the mRNA and protein expression of three key spine shapers: the actin-binding protein cyclase-associated protein 2 ( CAP2 ), the sheddase a disintegrin and metalloproteinase 10 ( ADAM10 ), and the synapse-associated protein 97 ( SAP97 ). Our analysis of the SCZ post-mortem brain indicated increased DLG1 mRNA in DLPFC and decreased CAP2 mRNA in the hippocampus of SCZ patients, compared to non-psychiatric control subjects, while the ADAM10 transcript was unaffected. Conversely, no differences in CAP2 , SAP97 , and ADAM10 protein levels were detected between SCZ and control individuals in both brain regions. To assess whether DLG1 and CAP2 transcript alterations were selective for SCZ, we also measured their expression in the superior frontal gyrus of patients affected by neurodegenerative disorders, like Parkinson's and Alzheimer's disease. Interestingly, also in Parkinson's disease patients, we found a selective reduction of CAP2 mRNA levels relative to controls but unaltered protein levels. Taken together, we reported for the first time altered CAP2 expression in the brain of patients with psychiatric and neurological disorders, thus suggesting that aberrant expression of this gene may contribute to synaptic dysfunction in these neuropathologies.

Background: Dysregulation of circular RNAs (circRNAs) plays an important role in the development of gastric cancer; thus, revealing the biological and molecular mechanisms of abnormally expressed circRNAs is critical for identifying novel therapeutic targets in gastric cancer., Methods: A circRNA microarray was performed to identify differentially expressed circRNAs between primary and distant metastatic tissues and between gastric cancer tissues sensitive or resistant to anti-programmed cell death 1 (PD-1) therapy. The expression of circRNA discs large homolog 1 (DLG1) was determined in a larger cohort of primary and distant metastatic gastric cancer tissues. The role of circDLG1 in gastric cancer progression was evaluated both in vivo and in vitro, and the effect of circDLG1 on the antitumor activity of anti-PD-1 was evaluated in vivo. The interaction between circDLG1 and miR-141-3p was assessed by RNA immunoprecipitation and luciferase assays., Results: circDLG1 was significantly upregulated in distant metastatic lesions and gastric cancer tissues resistant to anti-PD-1 therapy and was associated with an aggressive tumor phenotype and adverse prognosis in gastric cancer patients treated with anti-PD-1 therapy. Ectopic circDLG1 expression promoted the proliferation, migration, invasion, and immune evasion of gastric cancer cells. Mechanistically, circDLG1 interacted with miR-141-3p and acted as a miRNA sponge to increase the expression of CXCL12, which promoted gastric cancer progression and resistance to anti-PD-1-based therapy., Conclusions: Overall, our findings demonstrate how circDLG1 promotes gastric cancer cell proliferation, migration, invasion and immune evasion and provide a new perspective on the role of circRNAs during gastric cancer progression., (© 2021. The Author(s).)

CRAC, which plays important role in Ca 2+ -dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca 2+ -imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca 2+ -response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca 2+ -oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Zika Virus (ZIKV) is an RNA virus that belongs to the Flavivirus (FV) genus. In the last years, several unique characteristics of ZIKV among FV have been revealed, as the multiple routes of transmission and its ability to reach different human tissues, including the central nervous system. Thus, one of the most intriguing features of ZIKV biology is its ability to cross diverse complex biological barriers. The main aim of this study is to contribute to the understanding of the still unclear mechanisms behind this viral activity. We investigated an African strain and two South American ZIKV isolates belonging to the Asian lineage, in order to characterize possible differences regarding their ability to disturb intercellular junctions. The Asian isolates correspond to an imported (Venezuelan) and an autochthonous (Argentinian) ZIKV strain for which there is still no data available. We focused on occludin and DLG1 expression as markers of tight and adherent junctions, respectively. For this, we applied a quantitative immunofluorescence assay that can ascertain alterations in the cell junction proteins expression in the infected cells. Our findings indicated that the different ZIKV strains were able to reduce the levels of both polarity proteins without altering their overall cell distribution. Moreover, the grade of this effect was strain-dependent, being the DLG1 reduction higher for the African and Asian Venezuelan isolates and, on the contrary, occludin down-regulation was more noticeable for the Argentinian strain. Interestingly, among both junction proteins the viral infection caused a relative larger reduction in DLG1 expression for all viruses, suggesting DLG1 may be of particular relevance for ZIKV infections. Taken together, this study contributes to the knowledge of the biological mechanisms involved in ZIKV cytopathogenesis, with a special focus on regional isolates., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The retina is the innermost part of the eye of most vertebrates and it is essential for vision. The development, maintenance, and function of this laminated structure is tightly regulated by numerous genes. Deficiencies in the expression of these genes as well as deregulation of various molecular mechanisms can cause retinopathies and blindness. MicroRNAs (miRNAs) are one of the most important and effective molecular regulatory mechanisms that underlie the biology of the retina. miRNAs have specific functional roles in the development and maintenance of different retinal layers and retinal cell types. While previous studies have reported a large number of miRNAs linked to development, maintenance and diseases of the retina, no comprehensive study has properly discussed and integrated data from these studies. Given the particular importance of miR-204 in retinal biology, we intend to critically discuss the expression and functional significance of this miRNA in the development, maintenance, and pathologies of the retina. Moreover, we explore biological processes through which miR-204 influences retinal pathophysiology. This review highlights the crucial functions of miR-204 in the retina and suggests the putative mechanism of miR-204 action in retinal biology., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: Familial exudative vitreoretinopathy (FEVR) is a blinding retinal vascular disease. Clinically, FEVR is characterized by incomplete vascularization of the peripheral retina and pathological neovascularization. Only about 50% of FEVR cases can be explained by known FEVR disease gene variations. This study aimed to identify novel genes associated with the FEVR phenotype and explore their pathogenic mechanisms. Materials and Methods: Exome sequencing analyses were conducted on one Chinese family with FEVR whose affected members did not exhibit pathogenic variants in the known FEVR genes (verified using Sanger sequencing analysis). Functions of the affected proteins were evaluated using reporter assays. Western blot analysis was used to detect mutant protein expression and the genes' pathogenic mechanisms. Results: A rare novel heterozygous variant in DLG1 (c.1792A>G; p.S598G) was identified. The amino acid residues surrounding the identified variant are highly conserved among vertebrates. A luciferase reporter assay revealed that the mutant DLG1 protein DLG1 -S598G lost its ability to activate Wnt signaling. Moreover, a knockdown (KD) of DLG1 in human primary retinal endothelial cells impaired tube formation. Mechanistically, DLG1 KD led to a reduction in phosphorylated VEGFR2, an essential receptor for the angiogenic potency that signals the vascular endothelial growth factor molecule. Conclusions: The data reported here demonstrate that DLG1 is a novel candidate gene for FEVR.

We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in Mϕs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response., (©2020 Society for Leukocyte Biology.)

Rationale: Cardiotoxic β 1 adrenergic receptor (β 1 AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of β 1 AR and organizes a receptor signalosome., Objective: We aim to elucidate the dynamics of β 1 AR-SAP97 signalosome and its potential role in chronic cardiotoxic β 1 AR-CaMKII signaling that contributes to development of heart failure., Methods and Results: The integrity of cardiac β 1 AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine β 1 AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the β 1 AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of β 1 AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from β 1 AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of β 1 AR-SAP97 complex and increases in CaMKII activity in hearts., Conclusions: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac β 1 AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Background and Methods: Based on our previous discovery that SAP97 rs3915512 polymorphism significantly affects the cognitive function of schizophrenia, we further genotyped the other 12 single-nucleotide polymorphisms (SNPs) capturing the known common haplotype variations of this gene in a sample including 1014 patients with schizophrenia and 1078 matched controls., Results: There were no significant differences in the distribution of genotypes and alleles of the 12 SNPs of SAP97 between the patients and the controls (all P > 0.05). But, in the evaluation of the phenotypic effects of these SNPs on the patients' clinical symptoms and cognitive functions. While patients with minor allele in the rs9843659 polymorphism had higher N5 (difficulty in abstract thinking) scores than that with the main genotype (P = 0.002, Pcor = 0.014), the patients with minor allele in the rs6805920, rs4916461 and rs7638423 had lower verbal memory scores (P = 0.003, 0.003, 0.001, Pcor = 0.021, 0.021, 0.007, respectively) and the P values of these SNPs were still significant after the Bonferroni correction., Conclusion: Our data are further to indicate that the SAP97 gene polymorphisms may affect neurocognitive function especially verbal memory and the first to suggest that the SAP97 rs9843659 polymorphism may influence abstract thinking of schizophrenic patients in the southern Han Chinese population.

Synapse-Associated Protein 97 (SAP97) is an anchoring protein that in cardiomyocytes targets to the membrane and regulates Na + and K + channels. Here we compared the electrophysiological effects of native (WT) and p.P888L SAP97, a common polymorphism. Currents were recorded in cardiomyocytes from mice trans-expressing human WT or p.P888L SAP97 and in Chinese hamster ovary (CHO)-transfected cells. The duration of the action potentials and the QT interval were significantly shorter in p.P888L-SAP97 than in WT-SAP97 mice. Compared to WT, p.P888L SAP97 significantly increased the charge of the Ca-independent transient outward (I to,f ) current in cardiomyocytes and the charge crossing Kv4.3 channels in CHO cells by slowing Kv4.3 inactivation kinetics. Silencing or inhibiting Ca/calmodulin kinase II (CaMKII) abolished the p.P888L-induced Kv4.3 charge increase, which was also precluded in channels (p.S550A Kv4.3) in which the CaMKII-phosphorylation is prevented. Computational protein-protein docking predicted that p.P888L SAP97 is more likely to form a complex with CaMKII than WT. The Na + current and the current generated by Kv1.5 channels increased similarly in WT-SAP97 and p.P888L-SAP97 cardiomyocytes, while the inward rectifier current increased in WT-SAP97 but not in p.P888L-SAP97 cardiomyocytes. The p.P888L SAP97 polymorphism increases the I to,f , a CaMKII-dependent effect that may increase the risk of arrhythmias.

Synapse-associated protein 97 (SAP97) is a scaffolding protein crucial for the functional expression of several cardiac ion channels and therefore proper cardiac excitability. Alterations in the functional expression of SAP97 can modify the ionic currents underlying the cardiac action potential and consequently confer susceptibility for arrhythmogenesis. In this study, we generated a murine model for inducible, cardiac-targeted Sap97 ablation to investigate arrhythmia susceptibility and the underlying molecular mechanisms. Furthermore, we sought to identify human SAP97 ( DLG1 ) variants that were associated with inherited arrhythmogenic disease. The murine model of cardiac-specific Sap97 ablation demonstrated several ECG abnormalities, pronounced action potential prolongation subject to high incidence of arrhythmogenic afterdepolarizations and notable alterations in the activity of the main cardiac ion channels. However, no DLG1 mutations were found in 40 unrelated cases of genetically elusive long QT syndrome (LQTS). Instead, we provide the first evidence implicating a gain of function in human DLG1 mutation resulting in an increase in Kv4.3 current ( I to ) as a novel, potentially pathogenic substrate for Brugada syndrome (BrS). In conclusion, DLG1 joins a growing list of genes encoding ion channel interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. Dysfunction in these critical components of cardiac excitability can potentially result in fatal cardiac disease. NEW & NOTEWORTHY The gene encoding SAP97 ( DLG1 ) joins a growing list of genes encoding ion channel-interacting proteins (ChIPs) identified as potential channelopathy-susceptibility genes because of their ability to regulate the trafficking, targeting, and modulation of ion channels that are critical for the generation and propagation of the cardiac electrical impulse. In this study we provide the first data supporting DLG1- encoded SAP97's candidacy as a minor Brugada syndrome susceptibility gene.

Objective: To investigate the relationship between SAP97 genetic polymorphisms and sporadic Parkinson's disease (PD) in Han Chinese population with the expectation of offering genetic data for the early prevention and treatment of the disease., Methods: In this study, we genotyped single-nucleotide polymorphisms (SNPs) (rs3915512 and rs9843659) in theSAP97 gene in 317 patients with PD and 317 healthy-matched controls in a Han Chinese population through the improved multiplex ligation detection reaction (imLDR) technique. Then, we analyzed the association of each SNP, alone or in combination, with risk or age of onset of PD., Results: The SAP97 rs3915512 and rs9843659 polymorphisms were not associated with the risk of PD. However, the minor allele of the rs3915512 and rs9843659 were significantly more common in PD patients with an early age of onset. Additionally, significant differences in the distribution of the onset age of the PD among different genotypes of the rs9843659 polymorphism. The CA haplotype was significantly related to early onset PD., Conclusions: Our data are the first to suggest that the SAP97 SNPs rs3915512 and rs9843659 and the CA haplotype may be significantly associated with early onset PD in China., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020. Published by Elsevier B.V.)

Background: Persistent infection with high-risk Human Papillomavirus (HPVs) is associated with the development of cervical cancer. The transforming capacity of these viruses relies on the cooperative action of the E6 and E7 viral oncoproteins. Among the oncogenic activities of E6, the interaction and interference with cell polarity PDZ proteins have been well established. One of the most characterized PDZ targets of HPV E6 is human Disc large 1 (DLG1), a scaffolding protein involved in the control of cell polarity and proliferation. Interestingly, in cervical squamous intraepithelial lesions, alterations in DLG1 expression were observed in association to tumour progression. Moreover, the expression of both HPV E6 and E7 proteins may be responsible for the changes in DLG1 abundance and cell localization observed in the HPV-associated lesions., Methods: Due to the relevance of DLG1 deregulation in tumour development, we have performed an in-depth investigation of the expression of DLG1 in the presence of the HPV oncoproteins in epithelial cultured cells. The effects of HPV E6 and E7 proteins on DLG1 abundance and subcellular localization were assessed by western blot and confocal fluorescence microscopy, respectively., Results: We demonstrated that the relative abundance of HPV-18 E6 and DLG1 is a key factor that contributes to defining the expression abundance of both proteins. We also show here that a high expression level of DLG1 may negatively affect HPV-18 E6 nuclear expression. Moreover, the co-expression of HPV-18 E6 and E7 produces a striking effect on DLG1 subcellular localization and a co-distribution in the cytoplasmic region. Interestingly, HPV-18 E7 is also able to increase DLG1 levels, likely by rescuing it from the E6-mediated proteasomal degradation., Conclusions: In general, the data suggest that HPV-18 E6 and E7 may have opposing activities in regards to the regulation of DLG1 levels and may cooperatively contribute to its subcellular redistribution in the HPV context. These findings constitute a step forward in understanding the differential expression of DLG1 during tumour progression in an HPV-associated model.

Background: Benign thyroid nodules (BTN) are frequently diagnosed as papillary thyroid carcinoma (PTC), leading to unnecessary treatment. We found that plasma lncRNA DLG1-AS1 was upregulated in PTC patients but not in BTN patients and healthy controls., Methods: In this study DLG1-AS1 and miR-199a-3p in plasma of both PTC patients and BTN patients were detected by qPCR. ROC curve analysis was performed for diagnostic analysis. Overexpression experiments were performed to analyze the interaction between DLG1-AS1 and miR-199a-3p. CCK-8 assay was performed to analyze cell proliferation., Results: In this study, upregulation of DLG1-AS1 distinguished PTC patients from BTN patients and healthy controls. Plasma miR-199a-3p was downregulated in PTC patients compared with healthy controls and BTN patients. Plasma levels of miR-199a-3p were inversely correlated in PTC patients, but not in BTN patients and healthy controls. miR-199a-3p overexpression failed to significantly affect DLG1-AS1, while DLG1-AS1 overexpression resulted in downregulated miR-199a-3p, In addition, DLG1-AS1 overexpression promoted the proliferation of PTC cells. miR-199a-3p overexpression played an opposite role and attenuated the effects of DLG1-AS1 overexpression., Conclusions: Therefore, DLG1-AS1 may promote PTC by downregulating miR-199a-3p.

Oral clefts are composed of cleft of the lip, cleft of the lip and palate, or cleft of the palate, and they are associated with a wide range of expression and severity. When cleft of the palate is associated with cleft of the lip with preservation of the primary palate, it defines an atypical phenotype called discontinuous cleft. Although this phenotype may represent 5% of clefts of the lip and/or palate (CLP), it is rarely specifically referred to and its pathophysiology is unknown. We conducted whole exome sequencing (WES) and apply a candidate gene approach to non-syndromic discontinuous CLP individuals in order to identify genes and deleterious variants that could underlie this phenotype. We discovered loss-of-function variants in two out of the seven individuals, implicating FGFR1 and DLG1 genes, which represents almost one third of this cohort. Whole exome sequencing of clinically well-defined subgroups of CLP, such as discontinuous cleft, is a relevant approach to study CLP etiopathogenesis. It could facilitate more accurate clinical, epidemiological and fundamental research, ultimately resulting in better diagnosis and care of CLP patients. Non-syndromic discontinuous cleft lip and palate seems to have a strong genetic basis.

Background: Genetic risk variants in the hemizygous allele may influence neuropsychiatric manifestations and clinical course in 3q29 deletion carriers., Methods: In-depth phenotypic assessment in two deletion carriers included medical records, medical, genetic, psychiatric and neuropsychological evaluations, brain MRI scan and EEG. Blood samples were analyzed for copy number variations, and deep sequencing of the affected 3q29 region was performed in patients and seven first-degree relatives. Risk variants were identified through bioinformatic analysis., Results: One deletion carrier was diagnosed with learning difficulties and childhood autism, the other with mild intellectual disability and schizophrenia. EEG abnormalities in childhood normalized in adulthood in both. Cognitive abilities improved during adolescence in one deletion carrier. Both had microcytic, hypochromic erythrocytes and suffered from chronic pain and fatigue. Molecular and bioinformatic analyses identified risk variants in the hemizygous allele that were not present in the homozygous state in relatives in genes involved in cilia function and insulin action in the autistic individual and in synaptic function and neurosteroid transport in the subject with schizophrenia., Conclusion: 3q29 deletion carriers may undergo developmental phenotypic transition and need regular medical follow-up. Identified risk variants in the remaining hemizygous allele should be explored further in autism and schizophrenia research., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

Arginine-vasopressin (AVP)-mediated translocation of aquaporin-2 (AQP2) protein-forming water channels from storage vesicles to the membrane of renal collecting ducts is critical for the renal conservation of water. The type-1 PDZ-binding motif (PBM) in AQP2, "GTKA," is a critical barcode for its translocation, but its precise role and that of its interacting protein partners in this process remain obscure. We determined that synapse-associated protein-97 (SAP97), a membrane-associated guanylate kinase protein involved in establishing epithelial cell polarity, was an avid binding partner to the PBM of AQP2. The role of PBM and SAP97 on AQP2 redistribution in response to AVP was assessed in LLC-PK 1 renal collecting cells by confocal microscopy and cell surface biotinylation techniques. These experiments indicated that distribution of AQP2 and SAP97 overlapped in the kidneys and LLC-PK 1 cells and that knockdown of SAP97 inhibited the translocation of AQP2 in response to AVP. Binding between AQP2 and SAP97 was mediated by specific interactions between the second PDZ of SAP97 and PBM of AQP2. Mechanistically, inactivation of the PBM of AQP2, global delocalization of PKA, or knockdown of SAP97 inhibited AQP2 translocation as well as AVP- and forskolin-mediated phosphorylation of Ser 256 in AQP2, which serves as the major translocation barcode of AQP2. These results suggest that the targeting of PKA to the microdomain of AQP2 via SAP97-AQP2 interactions in association with cross-talk between two barcodes in AQP2, namely, the PBM and phospho-Ser 256 , plays an important role in the translocation of AQP2 in the kidney.

Dendritic cells (DCs) play key roles in Ab responses by presenting Ags to lymphocytes and by producing proinflammatory cytokines. In this study, we reported that DC-specific knockout of discs large homologue 1 (Dlg1) resulted in a significantly reduced capacity to mediate Ab responses to both thymus-independent and thymus-dependent Ags in Dlg1 fl/fl Cd11c-Cre-GFP mice. Mechanistically, Dlg1-deficient DCs showed severely impaired endocytosis and phagocytosis capacities upon Ag exposure. In parallel, loss of Dlg1 significantly jeopardized the proinflammatory cytokine production by DCs upon TLR stimulation. Thus, Dlg1-deficient DCs lost their functions to support innate and adaptive immunities. At a cellular level, Dlg1 exhibited an indispensable function to maintain membrane potential changes by securing potassium ion (K + ) efflux and subsequent calcium ion (Ca 2+ ) influx events in DCs upon stimulation, both of which are known to be required for proper function of DCs. At a molecular level, Dlg1 did so by retaining the integrity of voltage-gated K + channels (including Kv1.3) in DCs. The loss of Dlg1 led to a decreased expression of K + channels, resulting in impaired membrane potential changes and, as a consequence, reduced proinflammatory cytokine production, compromised Ag endocytosis, and phagocytosis. In conclusion, this study provided, to our knowledge, a novel insight into Dlg1 and the voltage-gated K + channels axis in DC functions., (Copyright © 2019 by The American Association of Immunologists, Inc.)

Human disc large (DLG1) is a scaffolding protein that through the interaction with diverse cell partners participates in the control of key cellular processes such as polarity, proliferation and migration. Experimental data have mainly identified DLG1 as a tumor suppressor. An outstanding point for DLG1 protein is that altered DLG1 expression and DLG1 gene mutations were observed in different pathologies, including cancer and neurological and immunological disorders. Evident changes in DLG1 abundance and/or cell localization were identified in a number of studies suggesting its participation in molecular mechanisms responsible for the development of such illnesses. In this review, we focus on some of the latest findings regarding DLG1 alterations in different diseases as well as its potential use as a biomarker for pathological progression. We further address the current knowledge on the molecular mechanisms regulating DLG1 expression and the posttranslational modifications that may affect DLG1 cell localization and functions. Despite the advances in this field, there are still open questions about the precise molecular link between alterations in DLG1 expression and the development of each specific pathology. The complete understanding of this concern will give us new scenarios for the design of promising diagnosis and therapeutic tools.

Both alterations to the epigenome and loss of polarity have been linked to cancer initiation, progression, and metastasis. It has previously been demonstrated that loss of the epigenetic reader protein Kaiso suppresses intestinal tumorigenesis in the Apc +/min mouse model, in which altered polarity plays a key role. Thus, we investigated the link between Kaiso deficiency, polarity, and suppression of intestinal tumorigenesis. We used Kaiso -deficient mice to conditionally delete Apc within the intestinal epithelia and demonstrated upregulation of the spindle polarity genes Dlg1 and Dlgap1 . To understand the role of Dlg1 , we generated Villin-creApc +/min Dlg1 flx/flx Kaiso -/y mice to analyze gene expression, survival, tumor burden, and spindle orientation. In vivo analysis of the Dlg1 -deficient intestine revealed improper orientation of mitotic spindles and a decreased rate of cellular migration. Loss of Dlg1 decreased survival in Apc +/min mice, validating its role as a tumor suppressor in the intestine. Significantly, the increased survival of Apc +/min Kaiso y/- mice was shown to be dependent on Dlg1 expression. Taken together, these data indicate that maintenance of spindle polarity in the intestinal crypt requires appropriate regulation of Dlg1 expression. As Dlg1 loss leads to incorrect spindle orientation and a delay in cells transiting the intestinal crypt. We propose that the delayed exit from the crypt increase the window in which spontaneous mutations can become fixed, producing a "tumor-permissive" environment, without an increase in mutation rate. IMPLICATIONS: Loss of mitotic spindle polarity delays the exit of cells from the intestinal crypt and promotes a tumorigenic environment., (©2018 American Association for Cancer Research.)

Synapse associated protein of 97KDa (SAP97) belongs to a family of scaffolding proteins, the membrane-associated guanylate kinases (MAGUKs), that are highly enriched in the postsynaptic density of synapses and play an important role in organizing protein complexes necessary for synaptic development and plasticity. The Dlg-MAGUK family of proteins are structurally very similar, and an effort has been made to parse apart the unique function of each Dlg-MAGUK protein by characterization of knockout mice. Knockout mice have been generated and characterized for PSD-95, PSD-93, and SAP102, however SAP97 knockout mice have been impossible to study because the SAP97 null mice die soon after birth due to a craniofacial defect. We studied the transcriptomic and behavioral consequences of a brain-specific conditional knockout of SAP97 (SAP97-cKO). RNA sequencing from hippocampi from control and SAP97-cKO male animals identified 67 SAP97 regulated transcripts. As large-scale genetic studies have implicated MAGUKs in neuropsychiatric disorders such as intellectual disability, autism spectrum disorders, and schizophrenia (SCZ), we analyzed our differentially expressed gene (DEG) set for enrichment of disease risk-associated genes, and found our DEG set to be specifically enriched for SCZ-related genes. Subjecting SAP97-cKO mice to a battery of behavioral tests revealed a subtle male-specific cognitive deficit and female-specific motor deficit, while other behaviors were largely unaffected. These data suggest that loss of SAP97 may have a modest contribution to organismal behavior. The SAP97-cKO mouse serves as a stepping stone for understanding the unique role of SAP97 in biology., Competing Interests: The authors have declared that no competing interests exist.

Mouse oocyte meiotic division requires the establishment of asymmetries in the oocyte before division, indicating the presence of polarity-establishing molecules. During mouse oocyte maturation proper orientation and positioning of the meiotic spindle at the oocyte cortex, as well as polarity in the oocyte cytoplasm and its oolemma, are necessary for the formation of functional haploid oocytes. Discs large homologue 1 (Dlg1) protein is a conserved protein that regulates cell polarity. In the present study, we found that Dlg1 was expressed at different stages of oocyte development. The localisation of Dlg1 during mouse oocyte maturation and its relationship with the cytoskeleton were analysed. Our data show that at the germinal vesicle stage, Dlg1 was present in the cytoplasm, prominently surrounding the germinal vesicle membrane. During maturation, Dlg1 became highly polarised by associating with the spindle and formed characteristic crescent-shaped accumulations under the cortex. Addition of nocodazole or cytochalasin B into the culture medium at different stages changed the localisation of Dlg1, indicating that the organisation of Dlg1 is a complex multi-step process and is dependent on microtubules and microfilaments. More importantly, we found that silencing of Dlg1 compromised the G2-M transition.