X-Linked Epilepsies: A Narrative Review.

X-linked epilepsies are a heterogeneous group of epileptic conditions, which often overlap with X-linked intellectual disability. To date, various X-linked genes responsible for epilepsy syndromes and/or developmental and epileptic encephalopathies have been recognized. The electro-clinical phenotype is well described for some genes in which epilepsy represents the core symptom, while less phenotypic details have been reported for other recently identified genes. In this review, we comprehensively describe the main features of both X-linked epileptic syndromes thoroughly characterized to date (PCDH19-related DEE, CDKL5-related DEE, MECP2-related disorders), forms of epilepsy related to X-linked neuronal migration disorders (e.g., ARX, DCX, FLNA) and DEEs associated with recently recognized genes (e.g., SLC9A6, SLC35A2, SYN1, ARHGEF9, ATP6AP2, IQSEC2, NEXMIF, PIGA, ALG13, FGF13, GRIA3, SMC1A). It is often difficult to suspect an X-linked mode of transmission in an epilepsy syndrome. Indeed, different models of X-linked inheritance and modifying factors, including epigenetic regulation and X-chromosome inactivation in females, may further complicate genotype-phenotype correlations. The purpose of this work is to provide an extensive and updated narrative review of X-linked epilepsies. This review could support clinicians in the genetic diagnosis and treatment of patients with epilepsy featuring X-linked inheritance.

OralExplorer: a web server for exploring the mechanisms of oral inflammatory diseases.

BACKGROUND: Oral inflammatory diseases are localized infectious diseases primarily caused by oral pathogens with the potential for serious systemic complications. However, publicly available datasets for these diseases are underutilized. To address this issue, a web tool called OralExplorer was developed. This tool integrates the available data and provides comprehensive online bioinformatic analysis. METHODS: Human oral inflammatory disease-related datasets were obtained from the GEO database and normalized using a standardized process. Transcriptome data were then subjected to differential gene expression analysis, immune infiltration analysis, correlation analysis, pathway enrichment analysis, and visualization. The single-cell sequencing data was visualized as cluster plot, feature plot, and heatmaps. The web platform was primarily built using Shiny. The biomarkers identified in OralExplorer were validated using local clinical samples through qPCR and IHC. RESULTS: A total of 35 human oral inflammatory disease-related datasets, covering 6 main disease types and 901 samples, were included in the study to identify potential molecular signatures of the mechanisms of oral diseases. OralExplorer consists of 5 main analysis modules (differential gene expression analysis, immune infiltration analysis, correlation analysis, pathway enrichment analysis and single-cell analysis), with multiple visualization options. The platform offers a simple and intuitive interface, high-quality images for visualization, and detailed analysis results tables for easy access by users. Six markers (IL1ß, SRGN, CXCR1, FGR, ARHGEF2, and PTAFR) were identified by OralExplorer. qPCR- and IHC-based experimental validation showed significantly higher levels of these genes in the periodontitis group. CONCLUSIONS: OralExplorer is a comprehensive analytical platform for oral inflammatory diseases. It allows users to interactively explore the molecular mechanisms underlying the action and regression of these diseases. It also aids dental researchers in unlocking the potential value of transcriptomics data related to oral diseases. OralExplorer can be accessed at https://smuonco.shinyapps.io/OralExplorer/  (Alternate URL: http://robinl-lab.com/OralExplorer ).

Dbl family RhoGEFs in cancer: different roles and targeting strategies.

Small Ras homologous guanosine triphosphatase (Rho GTPase) family proteins are highly associated with tumorigenesis and development. As intrinsic exchange activity regulators of Rho GTPases, Rho guanine nucleotide exchange factors (RhoGEFs) have been demonstrated to be closely involved in tumor development and received increasing attention. They mainly contain two families: the diffuse B-cell lymphoma (Dbl) family and the dedicator of cytokinesis (Dock) family. More and more emphasis has been paid to the Dbl family members for their abnormally high expression in various cancers and their correlation to poor prognosis. In this review, the common and distinctive structures of Dbl family members are discussed, and their roles in cancer are summarized with a focus on Ect2, Tiam1/2, P-Rex1/2, Vav1/2/3, Trio, KALRN, and LARG. Significantly, the strategies targeting Dbl family RhoGEFs are highlighted as novel therapeutic opportunities for cancer.

ARHGEF18 can promote BVDV NS5B activation of the host NF-κB signaling pathway by combining with the NS5B-palm domain.

Rho guanine nucleotide exchange factor 18 (ARHGEF18) is a member of the Rho guanine nucleotide exchange factor (RhoGEF) family. RhoGEF plays an important role in the occurrence of tumors and neurological diseases; however, its involvement in host cell resistance against pathogenic microorganisms is mostly unknown. Herein, we report that bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) can activate the nuclear factor kappa B (NF-κB) signaling pathway to induce an immune response. To clarify the functional domains of NS5B that activate NF-κB signaling, the six structural domains of NS5B were expressed separately: NS5B-core, NS5B-finger, NS5B-palm, NS5B-thumb, NS5B-N and NS5B-c domain. We preliminarily determined that the functional domains of NS5B that activate NF-κB signaling are the finger and palm domains. We used a bovine kidney cell cDNA library and yeast two-hybrid technology to identify that the host protein ARHGEF18 interacts with NS5B. Co-immunoprecipitation assays showed that ARHGEF18 interacts strongly with NS5B-palm. Interestingly ARHGEF18 could promote NF-κB signaling activation by BVDV NS5B. In addition silencing ARHGEF18 significantly inhibited NS5B-palm activation of NF-κB signaling. We concluded that ARHGEF18 can bind to BVDV NS5B through the palm domain to activate the NF-κB pathway. These findings provide direct evidence that BVDV NS5B induces immune responses by activating NF-κB signaling.

Advancements in the Research of GEF-H1: Biological Functions and Tumor Associations.

Guanine nucleotide exchange factor H1 (GEF-H1) is a unique protein modulated by the GDP/GTP exchange. As a regulator of the Rho-GTPase family, GEF-H1 can be activated through a microtubule-depended mechanism and phosphorylation regulation, enabling it to perform various pivotal biological functions across multiple cellular activities. These include the regulation of Rho-GTPase, cytoskeleton formation, cellular barrier, cell cycle, mitosis, cell differentiation, and vesicle trafficking. Recent studies have revealed its crucial effect on the tumor microenvironment (TME) components, promoting tumor initiation and progress. Consequently, an in-depth exploration of GEF-H1's biological roles and association with tumors holds promise for its potential as a valuable molecular target in tumor treatment.

Defining the Genetic Landscape of Congenital Mirror Movements in 80 Affected Individuals.

BACKGROUND: Congenital mirror movements (CMM) is a rare neurodevelopmental disorder characterized by involuntary movements from one side of the body that mirror voluntary movements on the opposite side. To date, five genes have been associated with CMM, namely DCC, RAD51, NTN1, ARHGEF7, and DNAL4. OBJECTIVE: The aim of this study is to characterize the genetic landscape of CMM in a large group of 80 affected individuals. METHODS: We screened 80 individuals with CMM from 43 families for pathogenic variants in CMM genes. In large CMM families, we tested for presence of pathogenic variants in multiple affected and unaffected individuals. In addition, we evaluated the impact of three missense DCC variants on binding between DCC and Netrin-1 in vitro. RESULTS: Causal pathogenic/likely pathogenic variants were found in 35% of probands overall, and 70% with familial CMM. The most common causal gene was DCC, responsible for 28% of CMM probands and 80% of solved cases. RAD51, NTN1, and ARHGEF7 were rare causes of CMM, responsible for 2% each. Penetrance of CMM in DCC pathogenic variant carriers was 68% and higher in males than females (74% vs. 54%). The three tested missense variants (p.Ile164Thr; p.Asn176Ser; and p.Arg1343His) bind Netrin-1 similarly to wild type DCC. CONCLUSIONS: A genetic etiology can be identified in one third of CMM individuals, with DCC being the most common gene involved. Two thirds of CMM individuals were unsolved, highlighting that CMM is genetically heterogeneous and other CMM genes are yet to be discovered. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Obscurin Maintains Myofiber Identity in Extraocular Muscles.

Purpose: The cytoskeleton of the extraocular muscles (EOMs) is significantly different from that of other muscles. We aimed to investigate the role of obscurin, a fundamental cytoskeletal protein, in the EOMs. Methods: The distribution of obscurin in human and zebrafish EOMs was compared using immunohistochemistry. The two obscurin genes in zebrafish, obscna and obscnb, were knocked out using CRISPR/Cas9, and the EOMs were investigated using immunohistochemistry, qPCR, and in situ hybridization. The optokinetic reflex (OKR) in five-day-old larvae and adult obscna-/-;obscnb-/- and sibling control zebrafish was analyzed. Swimming distance was recorded at the same age. Results: The obscurin distribution pattern was similar in human and zebrafish EOMs. The proportion of slow and fast myofibers was reduced in obscna-/-;obscnb-/- zebrafish EOMs but not in trunk muscle, whereas the number of myofibers containing cardiac myosin myh7 was significantly increased in EOMs of obscurin double mutants. Loss of obscurin resulted in less OKRs in zebrafish larvae but not in adult zebrafish. Conclusions: Obscurin expression is conserved in normal human and zebrafish EOMs. Loss of obscurin induces a myofiber type shift in the EOMs, with upregulation of cardiac myosin heavy chain, myh7, showing an adaptation strategy in EOMs. Our model will facilitate further studies in conditions related to obscurin.

Comparative interactome mapping of Tau-protein in classical and rapidly progressive Alzheimer's disease identifies subtype-specific pathways.

AIMS: Tau is a key player in Alzheimer's disease (AD) and other Tauopathies. Tau pathology in the brain directly correlates with neurodegeneration in AD. The recent identification of a rapid variant of AD demands an urgent need to uncover underlying mechanisms leading to differential progression in AD. Accordingly, we aimed to dissect the underlying differential mechanisms of toxicity associated with the Tau protein in AD subtypes and to find out subtype-dependent biomarkers and therapeutic targets. METHODS: To identify and characterise subtype-specific Tau-associated mechanisms of pathology, we performed comparative interactome mapping of Tau protein in classical AD (cAD) and rapidly progressive AD (rpAD) cases using co-immunoprecipitation coupled with quantitative mass spectrometry. The mass spectrometry data were extensively analysed using several bioinformatics approaches. RESULTS: The comparative interactome mapping of Tau protein revealed distinct and unique interactors (DPYSL4, ARHGEF2, TUBA4A and UQCRC2) in subtypes of AD. Interestingly, an analysis of the Tau-interacting proteins indicated enrichment of mitochondrial organisation processes, including negative regulation of mitochondrion organisation, mitochondrial outer membrane permeabilisation involved in programmed cell death, regulation of autophagy of mitochondrion and necroptotic processes, specifically in the rpAD interactome. While, in cAD, the top enriched processes were related to oxidation-reduction process, transport and monocarboxylic acid metabolism. CONCLUSIONS: Overall, our results provide a comprehensive map of Tau-interacting protein networks in a subtype-dependent manner and shed light on differential functions/pathways in AD subtypes. This comprehensive map of the Tau-interactome has provided subsets of disease-related proteins that can serve as novel biomarkers/biomarker panels and new drug targets.

Allosteric Activation of RhoA Complexed with p115-RhoGEF Deciphered by Conformational Dynamics.

The Ras homologue family member A (RhoA) is a member of the Rho family, a subgroup of the Ras superfamily. RhoA interacts with the 115 kDa guanine nucleotide exchange factor (p115-RhoGEF), which assists in activation and binding with downstream effectors. Here, we use molecular dynamics (MD) simulations and essential dynamics analysis of the inactive RhoA-GDP and active RhoA-GTP, when bound to p115-RhoGEF to decipher the mechanism of RhoA activation at the structural level. We observe that inactive RhoA-GDP maintains its position near the catalytic site on the Dbl homology (DH) domain of p115-RhoGEF through the interaction of its Switch I region with the DH domain. We further show that the active RhoA-GTP is engaged in more interactions with the p115-RhoGEF membrane-bound Pleckstrin homology (PH) domain as compared to RhoA-GDP. We hypothesize that the role of the interactions between the active RhoA-GTP and the PH domain is to help release it from the DH domain upon activation. Our results support this premise, and our simulations uncover the beginning of this process and provide structural details. They also point to allosteric communication pathways that take part in RhoA activation to promote and strengthen the interaction between the active RhoA-GTP and the PH domain. Allosteric regulation also occurs among other members of the Rho superfamily. Collectively, we suggest that in the activation process, the role of the RhoA-GTP interaction with the PH domain is to release RhoA-GTP from the DH domain after activation, making it available to downstream effectors.

CCDC88B interacts with RASAL3 and ARHGEF2 and regulates dendritic cell function in neuroinflammation and colitis.

CCDC88B is a risk factor for several chronic inflammatory diseases in humans and its inactivation causes a migratory defect in DCs in mice. CCDC88B belongs to a family of cytoskeleton-associated scaffold proteins that feature protein:protein interaction domains. Here, we identified the Rho/Rac Guanine Nucleotide Exchange Factor 2 (ARHGEF2) and the RAS Protein Activator Like 3 (RASAL3) as CCDC88B physical and functional interactors. Mice defective in Arhgef2 or Rasal3 show dampened neuroinflammation, and display altered cellular response and susceptibility to colitis; ARHGEF2 maps to a human Chromosome 1 locus associated with susceptibility to IBD. Arhgef2 and Rasal3 mutant DCs show altered migration and motility in vitro, causing either reduced (Arhgef2) or enhanced (Rasal3) migratory properties. The CCDC88B/RASAL3/ARHGEF2 complex appears to regulate DCs migration by modulating activation of RHOA, with ARHGEF2 and RASAL3 acting in opposite regulatory fashions, providing a molecular mechanism for the involvement of these proteins in DCs immune functions.

CircFN1 promotes acute myeloid leukemia cell proliferation and invasion but refrains apoptosis via miR-1294/ARHGEF10L axis.

Previous studies have proved circFN1 is highly expressed in acute myeloid leukemia (AML) patients and AML cell lines. This study aims to investigate the impact of circFN1 on AML and its mechanism. Via real-time quantitative PCR to detect circFN1, miR-1294, ARHGEF10L expressions in clinical plasma samples and AML cell lines, AML cells were cultured in vitro and transfected with si-circFN1, pcDNA3.1-circFN1, and si-ARHGEF10L, respectively, or co-transfected pcDNA3.1-circFN1 + miR-1294 mimic and pcDNA3.1-circFN1 + si-ARHGEF10L. Using dual luciferase reporter experiment to detect the relationship between circFN1 and miR-1294, as well as miR-1294 and ARHGEF10L. CCK-8 was used to detect cell proliferation, Transwell to cell invasion, TUNEL staining and flow cytometry to detect cell apoptosis, RT-qPCR to circFN1 RNA, miR-1294, and ARHGEF10L expression levels in HL-60 cells, and western blot to ARHGEF10L protein expression level in HL-60 cells. We found highly expressed circFN1 and ARHGEF10L, as well as low-expressed miR-1294 in AML patients and AML cell lines. In contrast to si-NC group, si-circFN1 group could signally inhibit HL-60 cell proliferation and migration, but promote cell apoptosis; compared with mimic NC group, miR-1294 mimic group could visually inhibit HL-60 cell proliferation and migration, but promote cell apoptosis. miR-1294 was the target of circFN1, and ARHGEF10L was the target of miR-1294. Over-expressing miR-1294 or silencing ARHGEF10L could signally inhibit circFN1 promoting HL-60 cell proliferation and migration and repressing cell apoptosis. circFN1 promotes proliferation and invasion of AML cell and represses cell apoptosis via regulating miR-1294/ARHGEF10L axis, which provides new insight for molecular targeted-treatment for AML.

The RhoGEF Trio is transported by microtubules and affects microtubule stability in migrating neural crest cells.

Directed cell migration requires a local fine-tuning of Rho GTPase activity to control protrusion formation, cell-cell contraction, and turnover of cellular adhesions. The Rho guanine nucleotide exchange factor (GEF) TRIO is ideally suited to control RhoGTPase activity because it combines two distinct catalytic domains to control Rac1 and RhoA activity in one molecule. However, at the cellular level, this molecular feature also requires a tight spatiotemporal control of TRIO activity. Here, we analyze the dynamic localization of Trio in Xenopus cranial neural crest (NC) cells, where we have recently shown that Trio is required for protrusion formation and migration. Using live cell imaging, we find that the GEF2 domain, but not the GEF1 domain of Trio, dynamically colocalizes with EB3 at microtubule plus-ends. Microtubule-mediated transport of Trio appears to be relevant for its function in NC migration, as a mutant GEF2 construct lacking the SxIP motif responsible for microtubule plus-end localization was significantly impaired in its ability to rescue the Trio loss-of-function phenotype compared to wild-type GEF2. Furthermore, by analyzing microtubule dynamics in migrating NC cells, we observed that loss of Trio function stabilized microtubules at cell-cell contact sites compared to controls, whereas they were destabilized at the leading edge of NC cells. Our data suggest that Trio is transported by microtubules to distinct subcellular locations where it has different functions in controlling microtubule stability, cell morphology, and cell-cell interaction during directed NC migration.

Novel OBSCN variants associated with a risk to exercise-intolerance and rhabdomyolysis.

Obscurin, encoded by the OBSCN gene, is a muscle protein consisting of three main splice isoforms, obscurin-A, obscurin-B, and obscurin kinase-only protein (also known as KIAA1639 or Obsc-kin). Obscurin is located at the M-band and Z-disks and interacts with titin and myomesin. It plays an important role in the stability and maintenance of the A- and M-bands and the subsarcolemmal organization of the microtubule network. Furthermore, obscurin is involved in Ca2+ regulation and sarcoplasmic reticulum function and is connected to several other muscle proteins. OBSCN gene variants have been reported to be relatively common in inherited cardiomyopathies. Here we reported two young patients with a history of cramps, myalgia, exercise intolerance, rhabdomyolysis, and myoglobinuria without any evidence of concomitant cardiomyopathy in association with novel OBSCN variants (c.24822C>A and c.2653+1G>C). Obscurin-deficient muscle fibers seem to have increased susceptibility to damage triggered by exercise that may lead to rhabdomyolysis. More studies are needed to clarify the diverse clinical phenotypes and the pathophysiology of OBSCN gene variants.

High placental expression of FLT1, LEP, PHYHIP and IL3RA - In persons of African ancestry with severe preeclampsia.

INTRODUCTION: Mortality from preeclampsia (PE) and PE-associated morbidities are 3-to 5-fold higher in persons of African ancestry than in those of Asian and European ancestries. METHODS: To elucidate placental contribution to worse PE outcomes in African ancestry pregnancies, we performed bulk RNA sequencing on 50 placentas from persons with severe PE (sPE) of African (n = 9), Asian (n = 18) and European (n = 23) ancestries and 73 normotensive controls of African (n = 10), Asian (n = 15) and European (n = 48) ancestries. RESULTS: Previously described canonical preeclampsia genes, involved in metabolism and hypoxia/angiogenesis including: LEP, HK2, FSTL3, FLT1, ENG, TMEM45A, ARHGEF4 and HTRA1 were upregulated sPE versus normotensive placentas across ancestries. LTF, NPR3 and PHYHIP were higher in African vs. Asian ancestry sPE placentas. Allograft rejection/adaptive immune response genes were upregulated in placentas from African but not in Asian or European ancestry sPE patients; IL3RA was of particular interest because the patient with the highest placental IL3RA expression, a person of African ancestry with sPE, developed postpartum cardiomyopathy, and was the only patient out of 123, that developed this condition. Interestingly, the sPE patients with the highest IL3RA expression among persons of Asian and European ancestries developed unexplained tachycardia peripartum, necessitating echocardiography in the European ancestry patient. The association between elevated placental IL3RA levels and unexplained tachycardia or peripartum cardiomyopathy was found to be significant in the 50 sPE patients (p = .0005). DISCUSSION: High placental upregulation of both canonical preeclampsia and allograft rejection/adaptive immune response genes may contribute to worse PE outcomes in African ancestry sPE patients.

Oncogenic Gαq activates RhoJ through PDZ-RhoGEF.

Oncogenic Gαq causes uveal melanoma via non-canonical signaling pathways. This constitutively active mutant GTPase is also found in cutaneous melanoma, lung adenocarcinoma, and seminoma, as well as in benign vascular tumors, such as congenital hemangiomas. We recently described that PDZ-RhoGEF (also known as ARHGEF11), a canonical Gα12/13 effector, is enabled by Gαs Q227L to activate CdcIn addition, and we demonstrated that constitutively active Gαq interacts with the PDZ-RhoGEF DH-PH catalytic module, but does not affect its binding to RhoA or Cdc. This suggests that it guides this RhoGEF to gain affinity for other GTPases. Since RhoJ, a small GTPase of the Cdc42 subfamily, has been involved in tumor-induced angiogenesis and the metastatic dissemination of cancer cells, we hypothesized that it might be a target of oncogenic Gαq signaling via PDZ-RhoGEF. Consistent with this possibility, we found that Gαq Q209L drives full-length PDZ-RhoGEF and a DH-PH construct to interact with nucleotide-free RhoJ-G33A, a mutant with affinity for active RhoJ-GEFs. Gαq Q209L binding to PDZ-RhoGEF was mapped to the PH domain, which, as an isolated construct, attenuated the interaction of this mutant GTPase with PDZ-RhoGEF's catalytic module (DH-PH domains). Expression of these catalytic domains caused contraction of endothelial cells and generated fine cell sprouts that were inhibited by co-expression of dominant negative RhoJ. Using relational data mining of uveal melanoma patient TCGA datasets, we got an insight into the signaling landscape that accompanies the Gαq/PDZ-RhoGEF/RhoJ axis. We identified three transcriptional signatures statistically linked with shorter patient survival, including GPCRs and signaling effectors that are recognized as vulnerabilities in cancer cell synthetic lethality datasets. In conclusion, we demonstrated that an oncogenic Gαq mutant enables the PDZ-RhoGEF DH-PH module to recognize RhoJ, suggesting an allosteric mechanism by which this constitutively active GTPase stimulates RhoJ via PDZ-RhoGEF. These findings highlight PDZ-RhoGEF and RhoJ as potential targets in tumors driven by mutant Gαq.

Ephexin3/ARHGEF5 Together with Cell Migration Signaling Partners within the Tumor Microenvironment Define Prognostic Transcriptional Signatures in Multiple Cancer Types.

Cancer cell migration involves a repertoire of signaling proteins that lead cytoskeleton reorganization as a critical step in metastatic dissemination. RhoGEFs are multidomain effectors that integrate signaling inputs to activate the molecular switches that orchestrate actin cytoskeleton reorganization. Ephexins, a group of five RhoGEFs, play oncogenic roles in invasive and metastatic cancer, leading to a mechanistic hypothesis about their function as signaling nodes assembling functional complexes that guide cancer cell migration. To identify clinically significant Ephexin signaling partners, we applied three systematic data mining strategies, based on the screening of essential Ephexins in multiple cancer cell lines and the identification of coexpressed signaling partners in the TCGA cancer patient datasets. Based on the domain architecture of encoded proteins and gene ontology criteria, we selected Ephexin signaling partners with a role in cytoskeletal reorganization and cell migration. We focused on Ephexin3/ARHGEF5, identified as an essential gene in multiple cancer cell types. Based on significant coexpression data and coessentiality, the signaling repertoire that accompanies Ephexin3 corresponded to three groups: pan-cancer, cancer-specific and coessential. To further select the Ephexin3 signaling partners likely to be relevant in clinical settings, we first identified those whose high expression was statistical linked to shorter patient survival. The resulting Ephexin3 transcriptional signatures represent significant accumulated risk, predictive of shorter survival, in 17 cancer types, including PAAD, LUAD, LGG, OSC, AML, KIRC, THYM, BLCA, LIHC and UCEC. The signaling landscape that accompanies Ephexin3 in various cancer types included the tyrosine kinase receptor MET and the tyrosine phosphatase receptor PTPRF, the serine/threonine kinases MARK2 and PAK6, the Rho GTPases RHOD, RHOF and RAC1, and the cytoskeletal regulator DIAHP1. Our findings set the basis to further explore the role of Ephexin3/ARHGEF5 as an essential effector and signaling hub in cancer cell migration.

Muscarinic receptor agonist-induced ßPix binding to ß-catenin promotes colon neoplasia.

M3 muscarinic receptors (M3R) modulate ß-catenin signaling and colon neoplasia. CDC42/RAC guanine nucleotide exchange factor, ßPix, binds to ß-catenin in colon cancer cells, augmenting ß-catenin transcriptional activity. Using in silico, in vitro, and in vivo approaches, we explored whether these actions are regulated by M3R. At the invasive fronts of murine and human colon cancers, we detected co-localized nuclear expression of ßPix and ß-catenin in stem cells overexpressing M3R. Using immunohistochemistry, immunoprecipitation, proximity ligand, and fluorescent cell sorting assays in human tissues and established and primary human colon cancer cell cultures, we detected time-dependent M3R agonist-induced cytoplasmic and nuclear association of ßPix with ß-catenin. ßPix knockdown attenuated M3R agonist-induced human colon cancer cell proliferation, migration, invasion, and expression of PTGS2, the gene encoding cyclooxygenase-2, a key player in colon neoplasia. Overexpressing ßPix dose-dependently augmented ß-catenin binding to the transcription factor TCF4. In a murine model of sporadic colon cancer, advanced neoplasia was attenuated in conditional knockout mice with intestinal epithelial cell deficiency of ßPix. Expression levels of ß-catenin target genes and proteins relevant to colon neoplasia, including c-Myc and Ptgs2, were reduced in colon tumors from ßPix-deficient conditional knockout mice. Targeting the M3R/ßPix/ß-catenin axis may have therapeutic potential.

A cross-species proteomic map reveals neoteny of human synapse development.

The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood1,2. Here we generate a cross-species proteomic map of synapse development in the human, macaque and mouse neocortex. By tracking the changes of more than 1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we find that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that human PSDs mature about two to three times slower than those of other species and contain higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of RhoGEF signalling in human neurons delays morphological maturation of dendritic spines and functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell-type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

Regulation of NLGN3 and the Synaptic Rho-GEF Signaling Pathway by CDK5.

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that are involved in synapse assembly and function. The NLGN gene family consists of 5 genes (NLGN1-3, 4X, and 4Y). NLGN3 forms heterodimers with other NLGNs and is expressed at both excitatory and inhibitory synapses, although the distinct role at different synapses is not fully understood. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase that targets various neuronal substrates to impact neuronal migration, neurite outgrowth, synaptic transmission, and plasticity. Both NLGNs and their presynaptic binding partners neurexins are highly associated with neurodevelopmental disorders. The NLGN3 gene is on the X chromosome and variants in NLGN3 have been linked to the pathophysiology in neurodevelopmental disorders. To better understand the endogenous modulation of NLGN3, we generated an HA-tagged knock-in mouse. We found that Cdk5 associates with NLGN3 in vivo and phosphorylates NLGN3 on serine 725 (S725) in the knock-in mouse of either sex. The phosphorylation affects the NLGN3 association with Kalirin-7, a postsynaptic guanine nucleotide exchange factors for Rho GTPase family proteins. We further observed that the phosphorylation modulates NLGN3 surface expression and NLGN3-mediated synaptic currents in cultured rat neurons. Thus, we characterized NLGN3 as a novel Cdk5 substrate and revealed the functional consequences of NLGN3 S725 phosphorylation in neurons. Our study provides a novel molecular mechanism underlying Cdk5-mediated regulation of postsynaptic cell adhesion molecules.SIGNIFICANCE STATEMENT NLGN3 is involved in synapse assembly and function at both excitatory and inhibitory synapses and has been associated with the pathophysiology of neurodevelopmental disorders. Cdk5 has brain-specific activity and is involved in neuronal transmission, synapse function, and plasticity. Here, we characterize NLGN3 as a Cdk5 substrate for the first time and show that Cdk5-mediated phosphorylation regulates NLGN3 function. We demonstrate that NLGN3 S725 is a Cdk5 phosphorylation site, and reveal that the site is important for NLGN3 association with Kalirin-7, NLGN3 surface expression, and NLGN3-mediated synaptic transmission.