Synapse Assembly Research Articles

Genomic basis of deep-water adaptation in Arctic Charr (Salvelinus alpinus) morphs.

The post-glacial colonization of Gander Lake in Newfoundland, Canada, by Arctic Charr (Salvelinus alpinus) provides the opportunity to study the genomic basis of adaptation to extreme deep-water environments. Colonization of deep-water (>50m) habitats often requires extensive adaptation to cope with novel environmental challenges from high hydrostatic pressure, low temperature, and low light, but the genomic mechanisms underlying evolution in these environments are rarely known. Here, we compare genomic divergence between a deep-water morph adapted to depths of up to 288m and a larger, piscivorous pelagic morph occupying shallower depths. Using both a SNP array and resequencing of whole nuclear and mitochondrial genomes, we find clear genetic divergence (FST =0.11-0.15) between deep and shallow water morphs, despite an absence of morph divergence across the mitochondrial genome. Outlier analyses identified many diverged genomic regions containing genes enriched for processes such as gene expression and DNA repair, cardiac function, and membrane transport. Detection of putative copy number variants (CNVs) uncovered 385genes with CNVs distinct to piscivorous morphs, and 275genes with CNVs distinct to deep-water morphs, enriched for processes associated with synapse assembly. Demographic analyses identified evidence for recent and local morph divergence, and ongoing reductions in diversity consistent with postglacial colonization. Together, these results show that Arctic Charr morph divergence has occurred through genome-wide differentiation and elevated divergence of genes underlying multiple cellular and physiological processes, providing insight into the genomic basis of adaptation in a deep-water habitat following postglacial recolonization.

Levels of SERPIN family proteins in peri-implant crevicular fluid in patients with peri-implantitis.

PurposeSerine protease inhibitors (SERPINs) family has been discovered in many disorders with proteolysis mechanisms. Our study determined the SERPINBs protein expression via public‐based GEO databases and further validated by peri‐implant crevicular fluid (PICF) of peri‐implantitis patients and healthy recruiters.MethodsThis study is a retrospective analysis. A total of 123 participants of Fujian Medical University Fujian Stomatological Hospital, consisting of 58 cases of peri‐implantitis and 65 samples of healthy control were retrospectively analyzed by ELISA assays and explored the gene enrichment pathways and clinical significance of SERPINBs expression accompanied by two different cytokines (IL‐6 and TNF‐α). Moreover, the clinical significance of SERPINBs was evaluated in peri‐implantitis patients with PICF samples by the receiver operating curve (ROC) using the area under the curve (AUC).ResultsKEGG database showed that Starch and sucrose metabolism, Retrograde endocannabinoid signaling, Prion diseases, Pentose phosphate pathways, and Olfactory pathways are up‐regulated; GO database showed that synapse organization, synapse assembly, sequestering of triglyceride, sensory perception of smell, and regulation of synapse organization pathways are up‐regulated. SERPINBs were overexpressed in peri‐implant tissues and peri‐implantitis patients with PICF. SERPINBs was positively correlated to IL‐6 and TNF‐α in peri‐implantitis patients with PICF. The ROC‐AUCs of SERPINBs achieved a significantly higher range from 0.895 to 0.939 in peri‐implantitis patients with PICF. Therefore, certain SERPINBs expressions were not only perceived through PICF and peri‐implant tissues but also showed potential significance in peri‐implantitis.ConclusionSERPINBs play an influential role in the pathogenesis of peri‐implantitis via binding with other inflammatory cytokines.

Upregulated NLGN1 predicts poor survival in colorectal cancer

BackgroundNeuroligin1 (NLGN1) is a main component of excitatory glutamatergic synapses complex and is important for synapse assembly and function. The clinical value of NLGN1 in colorectal cancer (CRC) is not clear.MethodsWe obtained the expression data of 1143 CRC patients from 3 independent Gene Expression Omnibus (GEO) datasets (GSE32323, GSE24551, GSE39582) and The Cancer Genome Atlas (TCGA) to make the comparison of the NLGN1 expression level between CRC tissues and matched noncancerous tissues, and to evaluate its value in predicting survival of CRC patients. At the protein level, these results were further confirmed by immunohistochemical staining of 52 CRC samples in our own centre. Finally, the function of NLGN1 was explored by gene set enrichment analysis (GSEA).ResultsIncreased mRNA and protein levels of NLGN1 expression were associated with worse overall survival or recurrence-free survival in CRC patients from 2 GEO datasets, the TCGA database, and our cohort. In addition, multivariate regression analysis showed that NLGN1 was an independent poor prognostic factor of survival in patients with CRC in TCGA database (OR = 2.524, P = 0.010). Functional analysis revealed that NLGN1 was correlated with function involving the Hedgehog signaling pathway, mismatch repair process, and some material metabolism processes.ConclusionsThis study is the first to implicate and verify NLGN1 as a new poor prognostic marker for CRC.

Increasing LFA-1 Expression Enhances Immune Synapse Architecture and T Cell Receptor Signaling in Jurkat E6.1 Cells.

The Jurkat E6.1 clone has been extensively used as a powerful tool for the genetic and biochemical dissection of the TCR signaling pathway. More recently, these cells have been exploited in imaging studies to identify key players in immunological synapse (IS) assembly in superantigen-specific conjugates and to track the dynamics of signaling molecules on glass surfaces coated with activating anti-CD3 antibodies. By comparison, Jurkat cells have been used only scantily for imaging on supported lipid bilayers (SLBs) incorporating laterally mobile TCR and integrin ligands, which allow to study synaptic rearrangements of surface molecules and the fine architecture of the mature IS, likely due to limitations in the assembly of immune synapses with well-defined architecture. Here we have explored whether upregulating the low levels of endogenous LFA-1 expression on Jurkat E6.1 cells through transduction with CD11a- and CD18-encoding lentiviruses can improve IS architecture. We show that, while forced LFA-1 expression did not affect TCR recruitment to the IS, E6.1 LFA-1high cells assembled better structured synapses, with a tighter distribution of signaling-competent TCRs at the center of the IS. LFA-1 upregulation enhanced protein phosphotyrosine signaling on SLBs but not at the IS formed in conjugates with SEE-pulsed APCs, and led to the constitutive formation of an intracellular phosphotyrosine pool co-localizing with endosomal CD3ζ. This was paralleled by an increase in the levels of p-ZAP-70 and p-Erk both under basal conditions and following activation, and in enhanced Ca2+ mobilization from intracellular stores. The enhancement in early signaling E6.1 LFA-1high cells did not affect expression of the early activation marker CD69 but led to an increase in IL-2 expression. Our results highlight a new role for LFA-1 in the core architecture of the IS that can be exploited to study the spatiotemporal redistribution of surface receptors on SLBs, thereby extending the potential of E6.1 cells and their derivatives for fine-scale imaging studies.

A CVID-associated variant in the ciliogenesis protein CCDC28B disrupts immune synapse assembly.

Ciliogenesis proteins orchestrate vesicular trafficking pathways that regulate immune synapse (IS) assembly in the non-ciliated T-cells. We hypothesized that ciliogenesis-related genes might be disease candidates for common variable immunodeficiency with impaired T-cell function (T-CVID). We identified a heterozygous, predicted pathogenic variant in the ciliogenesis protein CCDC28B present with increased frequency in a large CVID cohort. We show that CCDC28B participates in IS assembly by regulating polarized T-cell antigen receptor (TCR) recycling. This involves the CCDC28B-dependent, FAM21-mediated recruitment of the actin regulator WASH to retromer at early endosomes to promote actin polymerization. The CVID-associated CCDC28BR25W variant failed to interact with FAM21, leading to impaired synaptic TCR recycling. CVID T cells carrying the ccdc28b 211 C > T allele displayed IS defects mapping to this pathway that were corrected by overexpression of the wild-type allele. These results identify a new disease gene in T-CVID and pinpoint CCDC28B as a new player in IS assembly.

Morphological profiling of human T and NK lymphocytes by high-content cell imaging.

The immunological synapse is a complex structure that decodes stimulatory signals into adapted lymphocyte responses. It is a unique window to monitor lymphocyte activity because of development of systematic quantitative approaches. Here we demonstrate the applicability of high-content imaging to human T and natural killer (NK) cells and develop a pipeline for unbiased analysis of high-definition morphological profiles. Our approach reveals how distinct facets of actin cytoskeleton remodeling shape immunological synapse architecture and affect lytic granule positioning. Morphological profiling of CD8+ Tcells from immunodeficient individuals allows discrimination of the roles of the ARP2/3 subunit ARPC1B and the ARP2/3 activator Wiskott-Aldrich syndrome protein (WASP) in immunological synapse assembly. Single-cell analysis further identifies uncoupling of lytic granules and F-actin radial distribution in ARPC1B-deficient lymphocytes. Our study provides a foundation for development of morphological profiling as a scalable approach to monitor primary lymphocyte responsiveness and to identify complex aspects of lymphocyte micro-architecture.

F-Actin Dynamics in the Regulation of Endosomal Recycling and Immune Synapse Assembly.

Membrane proteins endocytosed at the cell surface as vesicular cargoes are sorted at early endosomes for delivery to lysosomes for degradation or alternatively recycled to different cellular destinations. Cargo recycling is orchestrated by multimolecular complexes that include the retromer, retriever, and the WASH complex, which promote the polymerization of new actin filaments at early endosomes. These endosomal actin pools play a key role at different steps of the recycling process, from cargo segregation to specific endosomal subdomains to the generation and mobility of tubulo-vesicular transport carriers. Local F-actin pools also participate in the complex redistribution of endomembranes and organelles that leads to the acquisition of cell polarity. Here, we will present an overview of the contribution of endosomal F-actin to T-cell polarization during assembly of the immune synapse, a specialized membrane domain that T cells form at the contact with cognate antigen-presenting cells.

Gene Dosage- and Age-Dependent Differential Transcriptomic Changes in the Prefrontal Cortex of Shank2-Mutant Mice.

Shank2 is an abundant postsynaptic scaffolding protein that is known to regulate excitatory synapse assembly and synaptic transmission and has been implicated in various neurodevelopmental disorders, including autism spectrum disorders (ASD). Previous studies on Shank2-mutant mice provided mechanistic insights into their autistic-like phenotypes, but it remains unclear how transcriptomic patterns are changed in brain regions of the mutant mice in age- and gene dosage-dependent manners. To this end, we performed RNA-Seq analyses of the transcripts from the prefrontal cortex (PFC) of heterozygous and homozygous Shank2-mutant mice lacking exons 6 and 7 at juvenile (week 3) and adult (week 12) stages. Juvenile heterozygous Shank2-mutant mice showed upregulation of glutamate synapse-related genes, downregulation of ribosomal and mitochondrial genes, and transcriptomic changes that are opposite to those observed in ASD (anti-ASD) such as upregulation of ASD_down (downregulated in ASD), GABA neuron-related, and oligodendrocyte-related genes. Juvenile homozygous Shank2 mice showed upregulation of chromatin-related genes and transcriptomic changes that are in line with those occurring in ASD (pro-ASD) such as downregulation of ASD_down, GABA neuron-related, and oligodendrocyte-related genes. Adult heterozygous and homozygous Shank2-mutant mice both exhibited downregulation of ribosomal and mitochondrial genes and pro-ASD transcriptomic changes. Therefore, the gene dosage- and age-dependent effects of Shank2 deletions in mice include differential transcriptomic changes across distinct functional contexts, including synapses, chromatin, ribosomes, mitochondria, GABA neurons, and oligodendrocytes.

The cell biology of synapse formation.

In a neural circuit, synapses transfer information rapidly between neurons and transform this information during transfer. The diverse computational properties of synapses are shaped by the interactions between pre- and postsynaptic neurons. How synapses are assembled to form a neural circuit, and how the specificity of synaptic connections is achieved, is largely unknown. Here, I posit that synaptic adhesion molecules (SAMs) organize synapse formation. Diverse SAMs collaborate to achieve the astounding specificity and plasticity of synapses, with each SAM contributing different facets. In orchestrating synapse assembly, SAMs likely act as signal transduction devices. Although many candidate SAMs are known, only a few SAMs appear to have a major impact on synapse formation. Thus, a limited set of collaborating SAMs likely suffices to account for synapse formation. Strikingly, several SAMs are genetically linked to neuropsychiatric disorders, suggesting that impairments in synapse assembly are instrumental in the pathogenesis of neuropsychiatric disorders.

ErbB4 promotes inhibitory synapse formation by cell adhesion, independent of its kinase activity

The precise control of the nervous system function under the vitality of synapses is extremely critical. Efforts have been taken to explore the underlying cellular and molecular mechanisms for synapse formation. Cell adhesion molecules have been found important for synapse assembly in the brain. Many trans-adhesion complexes have been identified to modulate excitatory synapse formation. However, little is known about the synaptogenic mechanisms for inhibitory synapses. ErbB4 is a receptor tyrosine kinase enriched in interneurons. Here, we showed that overexpressing ErbB4 in HEK293T cells induced gephyrin or GABAAR α1 puncta in co-cultured primary hippocampal neurons. This induction of ErbB4 was independent of its kinase activity. K751M, a kinase-dead mutant of ErbB4, can also induce gephyrin or GABAAR α1 puncta in the co-culture system. We further constructed K751M knock-in mice and found that the homozygous were viable at birth and fertile without changes in gross brain structure. The number of interneurons and inhibitory synapses onto pyramidal neurons (PyNs) were comparable between K751M and wild-type mice but decreased in ErbB4-Null mice. Moreover, ErbB4 can interact in trans with Slitrk3, a transmembrane postsynaptic protein at inhibitory synapses, through the extracellular RLD domain of ErbB4. The deletion of RLD diminished the induction of gephyrin or GABAAR α1 puncta by ErbB4. Finally, disruption of ErbB4–Slitrk3 interaction through neutralization of Slitrk3 by secretable RLD decreased inhibitory synapses onto PyNs and impaired GABAergic transmission. These results identify that ErbB4, as a cell adhesion molecule, promotes inhibitory synapse formation onto PyNs by interacting with Slitrk3 and in a kinase-independent manner, providing an unexpected mechanism of ErbB4 in inhibitory synapse formation.

Oligodendrocyte Secreted Factors Shape Hippocampal GABAergic Neuron Transcriptome and Physiology.

Oligodendrocytes form myelin for central nervous system axons and release factors which signal to neurons during myelination. Here, we ask how oligodendroglial factors influence hippocampal GABAergic neuron physiology. In mixed hippocampal cultures, GABAergic neurons fired action potentials (APs) of short duration and received high frequencies of excitatory synaptic events. In purified neuronal cultures without glial cells, GABAergic neuron excitability increased and the frequency of synaptic events decreased. These effects were largely reversed by adding oligodendrocyte conditioned medium (OCM). We compared the transcriptomic signature with the electrophysiological phenotype of single neurons in these three culture conditions. Genes expressed by single pyramidal or GABAergic neurons largely conformed to expected cell-type specific patterns. Multiple genes of GABAergic neurons were significantly downregulated by the transition from mixed cultures containing glial cells to purified neuronal cultures. Levels of these genes were restored by the addition of OCM to purified cultures. Clustering genes with similar changes in expression between different culture conditions revealed processes affected by oligodendroglial factors. Enriched genes are linked to roles in synapse assembly, AP generation, and transmembrane ion transport, including of zinc. These results provide new insight into the molecular targets by which oligodendrocytes influence neuron excitability and synaptic function.

Gene expression analysis of MCF7 cell lines of breast cancer treated with herbal extract of Cissampelos pareira revealed association with viral diseases

BackgroundIt is necessary to assess the cellular, molecular, and pathogenetic characteristics of COVID-19 and attention is required to understand highly effective gene targets and mechanisms. In this study, we suggest understandings into the fundamental pathogenesis of COVID-19 through gene expression analyses using the microarray data set GSE156445 publicly reachable at NIH/NCBI Gene Expression Omnibus database. The data set consists of MCF7 which is a human breast cancer cell line with estrogen, progesterone and glucocorticoid receptors. The cell lines treated with different quantities of Cissampelos pareira (Cipa). Cipa is a traditional medicinal plant which would possess an antiviral potency in preventing viral diseases such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. MethodsUtilizing Biobase, GEOquery, gplots packages in R studio, the differentially expressed genes (DEGs) were identified. The gene ontology (GO) of pathway enrichments employed by utilizing DAVID and KEGG enrichment analyses were studied. We further constructed a human protein-protein interaction (PPI) network and performed, based upon that, a subnetwork module analysis for significant signaling pathways. ResultsThe study identified 418 differentially expressed genes (DEGs) using bioinformatics tools. The gene ontology of pathway enrichments employed by GO and KEGG enrichment analyses of down-regulated and up-regulated DEGs were studied. Gene expression analysis utilizing gene ontology and KEGG results uncovered biological and signaling pathways such as “cell adhesion molecules”, “plasma membrane adhesion molecules”, “synapse assembly”, and “Interleukin-3-mediated signaling” which are mostly linked to COVID-19. Our results provide in silico evidence for candidate genes which are vital for the inhibition, adhesion, and encoding cytokine protein including LYN, IGFBP5, IL-1R1, and IL-13RA1 that may have strong biomarker potential for infectious diseases such as COVID-19 related therapy targets.

Downregulation of glial genes involved in synaptic function mitigates Huntington's disease pathogenesis.

Most research on neurodegenerative diseases has focused on neurons, yet glia help form and maintain the synapses whose loss is so prominent in these conditions. To investigate the contributions of glia to Huntington's disease (HD), we profiled the gene expression alterations of Drosophila expressing human mutant Huntingtin (mHTT) in either glia or neurons and compared these changes to what is observed in HD human and HD mice striata. A large portion of conserved genes are concordantly dysregulated across the three species; we tested these genes in a high-throughput behavioral assay and found that downregulation of genes involved in synapse assembly mitigated pathogenesis and behavioral deficits. To our surprise, reducing dNRXN3 function in glia was sufficient to improve the phenotype of flies expressing mHTT in neurons, suggesting that mHTT's toxic effects in glia ramify throughout the brain. This supports a model in which dampening synaptic function is protective because it attenuates the excitotoxicity that characterizes HD.

Intact synapse structure and function after combined knockout of PTPδ, PTPσ, and LAR.

It has long been proposed that leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cell-adhesion proteins that control synapse assembly. Their synaptic nanoscale localization, however, is not established, and synapse fine structure after knockout of the three vertebrate LAR-RPTPs (PTPδ, PTPσ, and LAR) has not been tested. Here, superresolution microscopy reveals that PTPδ localizes to the synaptic cleft precisely apposed to postsynaptic scaffolds of excitatory and inhibitory synapses. We next assessed synapse structure in newly generated triple-conditional-knockout mice for PTPδ, PTPσ, and LAR, complementing a recent independent study of synapse function after LAR-RPTP ablation (Sclip and Südhof, 2020). While mild effects on synaptic vesicle clustering and active zone architecture were detected, synapse numbers and their overall structure were unaffected, membrane anchoring of the active zone persisted, and vesicle docking and release were normal. Hence, despite their localization at synaptic appositions, LAR-RPTPs are dispensable for presynapse structure and function.

Latrophilin GPCR signaling mediates synapse formation.

Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In mouse CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo. Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.

The Role of the Cytoskeleton in Regulating the Natural Killer Cell Immune Response in Health and Disease: From Signaling Dynamics to Function.

Natural killer (NK) cells are innate lymphoid cells, which play key roles in elimination of virally infected and malignant cells. The balance between activating and inhibitory signals derived from NK surface receptors govern the NK cell immune response. The cytoskeleton facilitates most NK cell effector functions, such as motility, infiltration, conjugation with target cells, immunological synapse assembly, and cytotoxicity. Though many studies have characterized signaling pathways that promote actin reorganization in immune cells, it is not completely clear how particular cytoskeletal architectures at the immunological synapse promote effector functions, and how cytoskeletal dynamics impact downstream signaling pathways and activation. Moreover, pioneering studies employing advanced imaging techniques have only begun to uncover the architectural complexity dictating the NK cell activation threshold; it is becoming clear that a distinct organization of the cytoskeleton and signaling receptors at the NK immunological synapse plays a decisive role in activation and tolerance. Here, we review the roles of the actin cytoskeleton in NK cells. We focus on how actin dynamics impact cytolytic granule secretion, NK cell motility, and NK cell infiltration through tissues into inflammatory sites. We will also describe the additional cytoskeletal components, non-muscle Myosin II and microtubules that play pivotal roles in NK cell activity. Furthermore, special emphasis will be placed on the role of the cytoskeleton in assembly of immunological synapses, and how mutations or downregulation of cytoskeletal accessory proteins impact NK cell function in health and disease.

Single-Cell RNA Sequencing Analysis of the Drosophila Larval Ventral Cord.

Drosophila provides a powerful genetic system and an excellent model to study the development and function of the nervous system. The fly's small brain and complex behavior has been instrumental in mapping neuronal circuits and elucidating the neural basis of behavior. The fast pace of fly development and the wealth of genetic tools has enabled systematic studies on cell differentiation and fate specification, and has uncovered strategies for axon guidance and targeting. The accessibility of neuronal structures and the ability to edit and manipulate gene expression in selective cells and/or synaptic compartments has revealed mechanisms for synapse assembly and neuronal connectivity. Recent advances in single-cell RNA sequencing (scRNA-seq) have further enhanced our appreciation and understanding of neuronal diversity in a fly brain. However, due to the small size of the fly brain and its constituent cells, scRNA-seq methodologies require a few adaptations. Here, we describe a set of protocols optimized for scRNA-seq analysis of the Drosophila larval ventral nerve cord, starting from tissue dissection and cell dissociation to cDNA library preparation, sequencing, and data analysis. We apply this workflow to three separate samples and detail the technical challenges associated with successful application of scRNA-seq to studies on neuronal diversity. An accompanying article (Vicidomini, Nguyen, Choudhury, Brody, & Serpe, 2021) presents a custom multistage analysis pipeline that integrates modules contained in different R packages to ensure high-flexibility, high-quality RNA-seq data analysis. These protocols are developed for Drosophila larval ventral nerve cord, but could easily be adapted to other tissues and model organisms. © 2021 U.S. Government. Basic Protocol 1: Dissection of larval ventral nerve cords and preparation of single-cell suspensions Basic Protocol 2: Preparation and sequencing of single-cell transcriptome libraries Basic Protocol 3: Alignment of raw sequencing data to indexed genome and generation of count matrices.

Clustering Analysis Supports the Detection of Biological Processes Related to Autism Spectrum Disorder.

Genome sequencing has identified a large number of putative autism spectrum disorder (ASD) risk genes, revealing possible disrupted biological pathways; however, the genetic and environmental underpinnings of ASD remain mostly unanswered. The presented methodology aimed to identify genetically related clusters of ASD individuals. By using the VariCarta dataset, which contains data retrieved from 13,069 people with ASD, we compared patients pairwise to build “patient similarity matrices”. Hierarchical-agglomerative-clustering and heatmapping were performed, followed by enrichment analysis (EA). We analyzed whole-genome sequencing retrieved from 2062 individuals, and isolated 11,609 genetic variants shared by at least two people. The analysis yielded three clusters, composed, respectively, by 574 (27.8%), 507 (24.6%), and 650 (31.5%) individuals. Overall, 4187 variants (36.1%) were common to the three clusters. The EA revealed that the biological processes related to the shared genetic variants were mainly involved in neuron projection guidance and morphogenesis, cell junctions, synapse assembly, and in observational, imitative, and vocal learning. The study highlighted genetic networks, which were more frequent in a sample of people with ASD, compared to the overall population. We suggest that itemizing not only single variants, but also gene networks, might support ASD etiopathology research. Future work on larger databases will have to ascertain the reproducibility of this methodology.

Identification of Functional Genes in Pterygium Based on Bioinformatics Analysis.

Purpose The competing endogenous RNA (ceRNA) network regulatory has been investigated in the occurrence and development of many diseases. This research aimed at identifying the key RNAs of ceRNA network in pterygium and exploring the underlying molecular mechanism. Methods Differentially expressed long noncoding RNAs (lncRNAs), microRNAs (miRNAs), and mRNAs were obtained from the Gene Expression Omnibus (GEO) database and analyzed with the R programming language. LncRNA and miRNA expressions were extracted and pooled by the GEO database and compared with those in published literature. The lncRNA-miRNA-mRNA network was constructed of selected lncRNAs, miRNAs, and mRNAs. Metascape was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses on mRNAs of the ceRNA network and to perform Protein-Protein Interaction (PPI) Network analysis on the String website to find candidate hub genes. The Comparative Toxicogenomic Database (CTD) was used to find hub genes closely related to pterygium. The differential expressions of hub genes were verified using the reverse transcription-real-time fluorescent quantitative PCR (RT-qPCR). Result There were 8 lncRNAs, 12 miRNAs, and 94 mRNAs filtered to construct the primary ceRNA network. A key lncRNA LIN00472 ranking the top 1 node degree was selected to reconstruct the LIN00472 network. The GO and KEGG pathway enrichment showed the mRNAs in ceRNA networks mainly involved in homophilic cell adhesion via plasma membrane adhesion molecules, developmental growth, regulation of neuron projection development, cell maturation, synapse assembly, central nervous system neuron differentiation, and PID FOXM1 PATHWAY. According to the Protein-Protein Interaction Network (PPI) analysis on mRNAs in LINC00472 network, 10 candidate hub genes were identified according to node degree ranking. Using the CTD database, we identified 8 hub genes closely related to pterygium; RT-qPCR verified 6 of them were highly expressed in pterygium. Conclusion Our research found LINC00472 might regulate 8 hub miRNAs (miR-29b-3p, miR-183-5p, miR-138-5p, miR-211-5p, miR-221-3p, miR-218-5p, miR-642a-5p, miR-5000-3p) and 6 hub genes (CDH2, MYC, CCNB1, RELN, ERBB4, RB1) in the ceRNA network through mainly PID FOXM1 PATHWAY and play an important role in the development of pterygium.

The endosomal protein sorting nexin 4 is a synaptic protein

Sorting nexin 4 (SNX4) is an evolutionary conserved protein that mediates recycling from endosomes back to the plasma membrane in yeast and mammalian cells. SNX4 is expressed in the brain. Altered protein levels are associated with Alzheimer’s disease, but the neuronal localization and function of SNX4 have not been addressed. Using a new antibody, endogenous neuronal SNX4 co-localized with both early and recycling endosome markers, similar to the reported localization of SNX4 in non-neuronal cells. Neuronal SNX4 accumulated specifically in synaptic areas, with a predominant localization to presynaptic terminals. Acute depletion of neuronal SNX4 using independent short hairpin RNAs did not affect the levels of the transferrin receptor, a canonical SNX4 cargo. Quantitative mass spectrometry revealed that upon SNX4 knockdown the class of proteins involved in neurotransmission was the most dysregulated. This included integral membrane proteins at both the presynaptic and postsynaptic side of the synapse that participate in diverse synaptic processes such as synapse assembly, neurotransmission and the synaptic vesicle cycle. These data suggest that SNX4 is implicated in a variety of synaptic processes.