SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditis elegans.

The Polarity/Protusion model of UNC-6/Netrin function in axon repulsion does not rely on a gradient of UNC-6/Netrin. Instead, the UNC-5 receptor polarizes the VD growth cone such that filopodial protrusions are biased to the dorsal leading edge. UNC-5 then inhibits growth cone protrusion ventrally based upon this polarity, resulting in dorsally-biased protrusion and dorsal migration away from UNC-6/Netrin. While previous studies have shown that UNC-5 inhibits growth cone protrusion by destabilizing actin, preventing microtubule + end entry, and preventing vesicle fusion, the signaling pathways involved are unclear. The SRC-1 tyrosine kinase has been previously shown to physically interact with and phosphorylate UNC-5, and to act with UNC-5 in axon guidance and cell migration. Here, the role of SRC-1 in VD growth cone polarity and protrusion is investigated. A precise deletion of src-1 was generated, and mutants displayed unpolarized growth cones with increased size, similar to unc-5 mutants. Transgenic expression of src-1(+) in VD/DD neurons resulted in smaller growth cones, and rescued growth cone polarity defects of src-1 mutants, indicating cell-autonomous function. Transgenic expression of a putative kinase-dead src-1(D831A) mutant caused a phenotype similar to src-1 loss-of-function, suggesting that this is a dominant negative mutation. The D381A mutation was introduced into the endogenous src-1 gene by genome editing, which also had a dominant-negative effect. Genetic interactions of src-1 and unc-5 suggest they act in the same pathway on growth cone polarity and protrusion, but might have overlapping, parallel functions in other aspects of axon guidance. src-1 function was not required for the effects of activated myr::unc-5, suggesting that SRC-1 might be involved in UNC-5 dimerization and activation by UNC-6, of which myr::unc-5 is independent. In sum, these results show that SRC-1 acts with UNC-5 in growth cone polarity and inhibition of protrusion.

Long noncoding RNA UNC5B-AS1 suppresses cell proliferation by sponging miR-24-3p in glioblastoma multiforme.

BACKGROUND: Glioblastoma multiforme (GBM) is the most common primary CNS tumor, characterized by high mortality and heterogeneity. However, the related lncRNA signatures and their target microRNA (miRNA) for GBM are still mostly unknown. Therefore, it is critical that we discover lncRNA markers in GBM and their biological activities. MATERIALS AND METHODS: GBM-related RNA-seq data were obtained from the Cancer Genome Atlas (TCGA) database. The "edger" R package was used for differently expressed lncRNAs (DELs) identification. Then, we forecasted prospective miRNAs that might bind to lncRNAs by Cytoscape software. Survival analysis of those miRNAs was examined by the starBase database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the miRNAs' target genes was conducted by the Gene Set Enrichment Analysis (GSEA) database and R software. Moreover, the proliferative ability of unc-5 netrin receptor B antisense RNA 1 (UNC5B-AS1) cells was evaluated by Cell Counting Kit-8 (CCK-8) analysis. Mechanistically, the regulatory interaction between UNC5B-AS1 and miRNA in GBM biological processes was studied using CCK-8 analysis. RESULTS: Our results indicated that overexpression of UNC5B-AS1 has been shown to suppress GBM cell growth. Mechanistically, miR-24-3p in GBM was able to alleviate the anti-oncogenic effects of UNC5B-AS1 on cell proliferation. CONCLUSION: The discovery of the novel UNC5B-AS1-miR-24-3p network suggests possible lncRNA and miRNA roles in the development of GBM, which may have significant ramifications for the analysis of clinical prognosis and the development of GBM medications.

UNC5C: Novel Gene Associated with Psychiatric Disorders Impacts Dysregulation of Axon Guidance Pathways.

The UNC-5 family of netrin receptor genes, predominantly expressed in brain tissues, plays a pivotal role in various neuronal processes. Mutations in genes involved in axon development contribute to a wide spectrum of human diseases, including developmental, neuropsychiatric, and neurodegenerative disorders. The NTN1/DCC signaling pathway, interacting with UNC5C, plays a crucial role in central nervous system axon guidance and has been associated with psychiatric disorders during adolescence in humans. Whole-exome sequencing analysis unveiled two compound heterozygous causative mutations within the UNC5C gene in a patient diagnosed with psychiatric disorders. In silico analysis demonstrated that neither of the observed variants affected the allosteric linkage between UNC5C and NTN1. In fact, these mutations are located within crucial cytoplasmic domains, specifically ZU5 and the region required for the netrin-mediated axon repulsion of neuronal growth cones. These domains play a critical role in forming the supramodular protein structure and directly interact with microtubules, thereby ensuring the functionality of the axon repulsion process. We emphasize that these mutations disrupt the aforementioned processes, thereby associating the UNC5C gene with psychiatric disorders for the first time and expanding the number of genes related to psychiatric disorders. Further research is required to validate the correlation of the UNC5C gene with psychiatric disorders, but we suggest including it in the genetic analysis of patients with psychiatric disorders.

Counteracting Age-Related Netrin-1 Signaling Insufficiency Ameliorates Endothelial Cell Senescence and Angiogenesis Impairment.

Senescent cells that accumulate are regarded as promising therapeutic targets. However, senolytic therapy failed to achieve satisfactory results. We previously discovered that young human plasma improved vascular endothelial cell senescence, and UNC5B might be a novel intervention target. Netrin-1, as a natural ligand of UNC5B, plays roles in multiple age-related vascular disorders, but its involvement in aging is still unclear. Here, we observed a significant decrease in plasma Netrin-1 levels in old healthy subjects compared to the young. In vivo, adeno-associated-virus-mediated delivery of Netrin-1 into aged mice significantly improved functional recovery in a model of hindlimb ischemia, promoted angiogenesis in ischemic tissues, and activated the endothelial nitric oxide synthase. Furthermore, we revealed that low-dose Netrin-1 recombinant protein significantly reduced senescence-associated-ß-galactosidase-positive cells, inhibited the P53 pathway, promoted cell migration, increased tubule formation, and elevated nitric oxide production in senescent endothelial cells. However, UNC5B inhibition blocked the pro-angiogenesis effect of low-dose Netrin-1 on senescent cells or aortic rings. In summary, this study depicts that modulating Netrin-1 signaling can result in improved vascular health and Netrin-1 may have therapeutic potential for age-related ischemic diseases.

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

Fibronectin leucine-rich transmembrane protein 2 drives monocyte differentiation into macrophages via the UNC5B-Akt/mTOR axis.

Upon migrating into the tissues, hematopoietic stem cell (HSC)-derived monocytes differentiate into macrophages, playing a crucial role in determining innate immune responses towards external pathogens and internal stimuli. However, the regulatory mechanisms underlying monocyte-to-macrophage differentiation remain largely unexplored. Here we divulge a previously uncharacterized but essential role for an axon guidance molecule, fibronectin leucine-rich transmembrane protein 2 (FLRT2), in monocyte-to-macrophage maturation. FLRT2 is almost undetectable in human monocytic cell lines, human peripheral blood mononuclear cells (PBMCs), and mouse primary monocytes but significantly increases in fully differentiated macrophages. Myeloid-specific deletion of FLRT2 (Flrt2ΔMyel ) contributes to decreased peritoneal monocyte-to-macrophage generation in mice in vivo, accompanied by impaired macrophage functions. Gain- and loss-of-function studies support the promoting effect of FLRT2 on THP-1 cell and human PBMC differentiation into macrophages. Mechanistically, FLRT2 directly interacts with Unc-5 netrin receptor B (UNC5B) via its extracellular domain (ECD) and activates Akt/mTOR signaling. In vivo administration of mTOR agonist MYH1485 reverses the impaired phenotypes observed in Flrt2ΔMyel mice. Together, these results identify FLRT2 as a novel pivotal endogenous regulator of monocyte differentiation into macrophages. Targeting the FLRT2/UNC5B-Akt/mTOR axis may provide potential therapeutic strategies directly relevant to human diseases associated with aberrant monocyte/macrophage differentiation.

TOM-1/tomosyn acts with the UNC-6/netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans.

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.

EPB41L4A-AS1 and UNC5B-AS1 have diagnostic and prognostic significance in osteosarcoma.

BACKGROUND: Deregulation of lncRNAs has been observed in human osteosarcoma. This study explored the diagnostic and prognostic significance of EPB41L4A-AS1 and UNC5B-AS1 in osteosarcoma. METHODS: Relative levels of EPB41L4A-AS1 and UNC5B-AS1 were detected in osteosarcoma tissue samples and cells. The ability to distinguish osteosarcoma from health was assessed by receiver operating characteristic (ROC) curve construction. Kaplan-Meier (K-M) and Cox proportional-hazards analyses were performed for prognosis factors. The bioinformatics approach was used to identify targeting miRNA for EPB41L4A-AS1 and UNC5B-AS1. Kaplan-Meier survival curves and Whitney Mann U tests were conducted for validating the statistical significance. In cell culture experiments, the influence of EPB41L4A-AS1 and UNC5B-AS1 on proliferation, migration, and invasion of the osteosarcoma cell line was examined by CCK-8 and Transwell assays. RESULTS: Levels of EPB41L4A-AS1 and UNC5B-AS1 were upregulated in osteosarcoma patients and cells compared with the healthy participants and normal cell lines. EPB41L4A-AS1 and UNC5B-AS1 have a potent ability to distinguish the patients with osteosarcoma from the health. EPB41L4A-AS1 and UNC5B-AS1 levels correlated with SSS stage. Patients with high levels of EPB41L4A-AS1 and UNC5B-AS1 had significantly shorter survival times. EPB41L4A-AS1 and UNC5B-AS1 were independent prognostic indexes for overall survival. miR-1306-5p was a common target for EPB41L4A-AS1 and UNC5B-AS1. A propulsive impact on cell proliferation, migration, and invasion by EPB41L4A-AS1 and UNC5B-AS1 was observed, but can be rescued by miR-1306-5p. CONCLUSIONS: It was concluded that upregulations of EPB41L4A-AS1 and UNC5B-AS1 expression were diagnostic and prognostic biomarkers for human osteosarcoma. EPB41L4A-AS1 and UNC5B-AS1 contribute to the biological behavior of osteosarcoma via miR-1306-5p.

Axon targeting of Drosophila medulla projection neurons requires diffusible Netrin and is coordinated with neuroblast temporal patterning.

How axon guidance pathways are utilized in coordination with temporal and spatial patterning of neural progenitors to regulate neuropil assembly is not well understood. We study this question in the Drosophila medulla using the transmedullary (Tm) projection neurons that target lobula through the inner optic chiasm (IOC). We demonstrate that the Netrin pathway plays multiple roles in guidance of Tm axons and that temporal patterning of medulla neuroblasts determines pioneer versus follower Tm neurons during this process. Loss of Frazzled (Fra) in early-born pioneer Tm neurons leads to IOC defects, while loss of Fra from follower neurons does not affect the IOC. In the follower projection neurons, Fra is required in other targeting steps including lobula branch extension and layer-specific targeting. Furthermore, different from other identified scenarios of Netrin/Fra involved axon guidance in Drosophila, we demonstrate that diffusible Netrin is required for the correct axon targeting and optic lobe organization.

Notch-dependent binary fate choice regulates the Netrin pathway to control axon guidance of Drosophila visual projection neurons.

Notch-dependent binary fate choice between sister neurons is one of the mechanisms to generate neural diversity. How these upstream neural fate specification programs regulate downstream effector genes to control axon targeting and neuropil assembly remains less well understood. Here, we report that Notch-dependent binary fate choice in Drosophila medulla neurons is required to regulate the Netrin axon guidance pathway, which controls targeting of transmedullary (Tm) neurons to lobula. In medulla neurons of Notch-on hemilineage composed of mostly lobula-targeting neurons, Notch signaling is required to activate the expression of Netrin-B and repress the expression of its repulsive receptor Unc-5. Turning off Unc-5 is necessary for Tm neurons to target lobula. Furthermore, Netrin-B provided by Notch-on medulla neurons is required for correct targeting of Tm axons from later-generated medulla columns. Thus, the coordinate regulation of Netrin pathway components by Notch signaling ensures correct targeting of Tm axons and contributes to the neuropil assembly.

Netrins and Netrin Receptors are Essential for Normal Targeting of Sensory Axons in the Zebrafish Olfactory Bulb.

Olfactory sensory neurons that express related odorant receptors specifically target large identifiable neuropils called protoglomeruli when they first reach the olfactory bulb in the zebrafish. This crude odorant receptor-related mapping is further refined as odorant receptor-specific glomeruli segregate from protoglomeruli later in development. Netrins are a prominent class of axon guidance molecules whose contribution to olfactory circuit formation is poorly studied. Morpholino knock down experiments have suggested that Netrin/Dcc signaling is involved in normal protoglomerular targeting. Here we extend these findings with more detailed characterization and modeling of netrin expression, and by examining protoglomerular targeting in mutant lines fornetrin1a (ntn1a), netrin1b (ntn1b), and their receptorsunc5b,dcc, andneo1a. We confirm thatntn1a,ntn1b, anddccare required for normal protoglomerular guidance of a subset of olfactory sensory neurons that are labeled with the Tg(or111-7:IRES:Gal4) transgene. We also observe errors in the targeting of these axons inunc5bmutants, but not inneo1a mutants. Our findings are consistent with ntn1a andntn1bacting primarily as attractants for olfactory sensory neurons targeting the central zone protoglomerulus.

Expression of netrin and its receptors uncoordinated-5 and frazzled in arthropods and onychophorans suggests conserved and diverged functions in neuronal pathfinding and synaptogenesis.

BACKGROUND: Development of the nervous system and the correct connection of nerve cells require coordinated axonal pathfinding through an extracellular matrix. Outgrowing axons exhibit directional growth toward or away from external guidance cues such as Netrin. Guidance cues can be detected by growth cones that are located at the end of growing axons through membrane-bound receptors such as Uncoordianted-5 and Frazzled. Binding of Netrin causes reformation of the cytoskeleton and growth of the axon toward (or away from) the source of Netrin production. RESULTS: Here, we investigate the embryonic mRNA expression patterns of netrin genes and their potential receptors, uncoordinated-5 and frazzled in arthropod species that cover all main branches of Arthropoda, that is, Pancrustacea, Myriapoda, and Chelicerata. We also studied the expression patterns in a closely related outgroup species, the onychophoran Euperipatoides kanangrensis, and provide data on expression profiles of these genes in larval tissues of the fly Drosophila melanogaster including the brain and the imaginal disks. CONCLUSION: Our data reveal conserved and diverged aspects of neuronal guidance in Drosophila with respect to the other investigated species and suggest a conserved function in nervous system patterning of the developing appendages.

A Systematic Analysis of the Role of Unc-5 Netrin Receptor A (UNC5A) in Human Cancers.

Unc-5 netrin receptor A (UNC5A), a netrin family receptor, plays a key role in neuronal development and subsequent differentiation. Recently, studies have found that UNC5A plays an important role in multiple cancers, such as bladder cancer, non-small cell lung carcinoma, and colon cancer but its pan-cancer function is largely unknown. Herein, the R software and multiple databases or online websites (The Cancer Genome Atlas (TCGA), The Genotype-Tissue Expression (GTEx), The Tumor Immune Estimation Resource (TIMER), The Gene Set Cancer Analysis (GSCA), Gene Expression Profiling Interactive Analysis (GEPIA), and cBioPortal etc.) were utilized to examine the role of UNC5A in pan-cancer. UNC5A was found to be highly expressed across multiple human cancer tissues and cells, was linked to clinical outcomes of patients, and was a potential pan-cancer biomarker. The mutational landscape of UNC5A exhibited that patients with UNC5A mutations had poorer progress free survival (PFS) in head and neck squamous cell carcinoma (HNSC) and prostate adenocarcinoma (PRAD). Furthermore, UNC5A expression was associated with tumor mutation burden (TMB), neoantigen, tumor microenvironment (TME), tumor microsatellite instability (MSI), immunomodulators, immune infiltration, DNA methylation, immune checkpoint (ICP) genes, and drug responses. Our results suggest the potential of UNC5A as a pan-cancer biomarker and an efficient immunotherapy target, which may also guide drug selection for some specific cancer types in clinical practice.

GPC3-Unc5 receptor complex structure and role in cell migration.

Neural migration is a critical step during brain development that requires the interactions of cell-surface guidance receptors. Cancer cells often hijack these mechanisms to disseminate. Here, we reveal crystal structures of Uncoordinated-5 receptor D (Unc5D) in complex with morphogen receptor glypican-3 (GPC3), forming an octameric glycoprotein complex. In the complex, four Unc5D molecules pack into an antiparallel bundle, flanked by four GPC3 molecules. Central glycan-glycan interactions are formed by N-linked glycans emanating from GPC3 (N241 in human) and C-mannosylated tryptophans of the Unc5D thrombospondin-like domains. MD simulations, mass spectrometry and structure-based mutants validate the crystallographic data. Anti-GPC3 nanobodies enhance or weaken Unc5-GPC3 binding and, together with mutant proteins, show that Unc5/GPC3 guide migrating pyramidal neurons in the mouse cortex, and cancer cells in an embryonic xenograft neuroblastoma model. The results demonstrate a conserved structural mechanism of cell guidance, where finely balanced Unc5-GPC3 interactions regulate cell migration.

A novel signature to guide osteosarcoma prognosis and immune microenvironment: Cuproptosis-related lncRNA.

Objective: Osteosarcoma (OS) is a common bone malignancy with poor prognosis. We aimed to investigate the relationship between cuproptosis-related lncRNAs (CRLncs) and the survival outcomes of patients with OS. Methods: Transcriptome and clinical data of 86 patients with OS were downloaded from The Cancer Genome Atlas (TCGA). The GSE16088 dataset was downloaded from the Gene Expression Omnibus (GEO) database. The 10 cuproptosis-related genes (CRGs) were obtained from a recently published article on cuproptosis in Science. Combined analysis of OS transcriptome data and the GSE16088 dataset identified differentially expressed CRGs related to OS. Next, pathway enrichment analysis was performed. Co-expression analysis obtained CRLncs related to OS. Univariate COX regression analysis and least absolute shrinkage and selection operator (LASSO) regression analysis were used to construct the risk prognostic model of CRLncs. The samples were divided evenly into training and test groups to verify the accuracy of the model. Risk curve, survival, receiver operating characteristic (ROC) curve, and independent prognostic analyses were performed. Next, principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) analysis were performed. Single-sample gene set enrichment analysis (ssGSEA) was used to explore the correlation between the risk prognostic models and OS immune microenvironment. Drug sensitivity analysis identified drugs with potential efficacy in OS. Real-time quantitative PCR, Western blotting, and immunohistochemistry analyses verified the expression of CRGs in OS. Real-time quantitative PCR was used to verify the expression of CRLncs in OS. Results: Six CRLncs that can guide OS prognosis and immune microenvironment were obtained, including three high-risk CRLncs (AL645608.6, AL591767.1, and UNC5B-AS1) and three low-risk CRLncs (CARD8-AS1, AC098487.1, and AC005041.3). Immune cells such as B cells, macrophages, T-helper type 2 (Th2) cells, regulatory T cells (Treg), and immune functions such as APC co-inhibition, checkpoint, and T-cell co-inhibition were significantly downregulated in high-risk groups. In addition, we obtained four drugs with potential efficacy for OS: AUY922, bortezomib, lenalidomide, and Z.LLNle.CHO. The expression of LIPT1, DLAT, and FDX1 at both mRNA and protein levels was significantly elevated in OS cell lines compared with normal osteoblast hFOB1.19. The mRNA expression level of AL591767.1 was decreased in OS, and that of AL645608.6, CARD8-AS1, AC005041.3, AC098487.1, and UNC5B-AS1 was upregulated in OS. Conclusion: CRLncs that can guide OS prognosis and the immune microenvironment and drugs that may have a potential curative effect on OS obtained in this study provide a theoretical basis for OS survival research and clinical decision-making.

Somatotopy of Mouse Spinothalamic Innervation and the Localization of a Noxious Stimulus Requires Deleted in Colorectal Carcinoma Expression by Phox2a Neurons.

Anterolateral system (AS) neurons transmit pain signals from the spinal cord to the brain. Their morphology, anatomy, and physiological properties have been extensively characterized and suggest that specific AS neurons and their brain targets are concerned with the discriminatory aspects of noxious stimuli, such as their location or intensity, and their motivational/emotive dimension. Among the recently unraveled molecular markers of AS neurons is the developmentally expressed transcription factor Phox2a, providing us with the opportunity to selectively disrupt the embryonic wiring of AS neurons to gain insights into the logic of their adult function. As mice with a spinal-cord-specific loss of the netrin-1 receptor deleted in colorectal carcinoma (DCC) have increased AS neuron innervation of ipsilateral brain targets and defective noxious stimulus localization or topognosis, we generated mice of either sex carrying a deletion of Dcc in Phox2a neurons. Such DccPhox2a mice displayed impaired topognosis along the rostrocaudal axis but with little effect on left-right discrimination and normal aversive responses. Anatomical tracing experiments in DccPhox2a mice revealed defective targeting of cervical and lumbar AS axons within the thalamus. Furthermore, genetic labeling of AS axons revealed their expression of DCC on their arrival in the brain, at a time when many of their target neurons are being born and express Ntn1 Our experiments suggest a postcommissural crossing function for netrin-1:DCC signaling during the formation of somatotopically ordered maps and are consistent with a discriminatory function of some of the Phox2a AS neurons.SIGNIFICANCE STATEMENT How nociceptive (pain) signals are relayed from the body to the brain remains an important question relevant to our understanding of the basic physiology of pain perception. Previous studies have demonstrated that the AS is a main effector of this function. It is composed of AS neurons located in the spinal cord that receive signals from nociceptive sensory neurons that detect noxious stimuli. In this study, we generate a genetic miswiring of mouse AS neurons that results in a decreased ability to perceive the location of a painful stimulus. The precise nature of this defect sheds light on the function of different kinds of AS neurons and how pain information may be organized.

Netrin-1 induces the anti-apoptotic and pro-survival effects of B-ALL cells through the Unc5b-MAPK axis.

BACKGROUND: B-cell acute lymphoblastic leukemia (B-ALL) comprises over 85% of all acute lymphoblastic leukemia (ALL) cases and is the most common childhood malignancy. Although the 5 year overall survival of patients with B-ALL exceeds 90%, patients with relapsed or refractory B-ALL may suffer from poor prognosis and adverse events. The axon guidance factor netrin-1 has been reported to be involved in the tumorigenesis of many types of cancers. However, the impact of netrin-1 on B-ALL remains unknown. METHODS: The expression level of netrin-1 in peripheral blood samples of children with B-ALL and children without neoplasia was measured by enzyme-linked immunosorbent assay (ELISA) kits. Then, CCK-8 cell proliferation assays and flow cytometric analysis were performed to detect the viability and apoptosis of B-ALL cells (Reh and Sup B15) treated with exogenous recombinant netrin-1 at concentrations of 0, 25, 50, and 100 ng/ml. Furthermore, co-immunoprecipitation(co-IP) was performed to detect the receptor of netrin-1. UNC5B expression interference was induced in B-ALL cells with recombinant lentivirus, and then CCK-8 assays, flow cytometry assays and western blotting assays were performed to verify that netrin-1 might act on B-ALL cells via the receptor Unc5b. Finally, western blotting and kinase inhibitor treatment were applied to detect the downstream signaling pathway. RESULTS: Netrin-1 expression was increased in B-ALL, and netrin-1 expression was upregulated in patients with high- and intermediate-risk stratification group of patients. Then, we found that netrin-1 induced an anti-apoptotic effect in B-ALL cells, implying that netrin-1 plays an oncogenic role in B-ALL. co-IP results showed that netrin-1 interacted with the receptor Unc5b in B-ALL cells. Interference with UNC5B was performed in B-ALL cells and abolished the antiapoptotic effects of netrin-1. Further western blotting was applied to detect the phosphorylation levels of key molecules in common signaling transduction pathways in B-ALL cells treated with recombinant netrin-1, and the FAK-MAPK signaling pathway was found to be activated. The anti-apoptotic effect of netrin-1 and FAK-MAPK phosphorylation was abrogated by UNC5B interference. FAK inhibitor treatment and ERK inhibitor treatment were applied and verified that the FAK-MAPK pathway may be downstream of Unc5b. CONCLUSION: Taken together, our findings suggested that netrin-1 induced the anti-apoptotic effect of B-ALL cells through activation of the FAK-MAPK signaling pathway by binding to the receptor Unc5b. Video Abstract.

PTBP1 knockdown promotes neural differentiation of glioblastoma cells through UNC5B receptor.

Rationale: Cell reprogramming technology is utilized to prevent cancer progression by transforming cells into terminally differentiated, non-proliferating states. Polypyrimidine tract binding protein 1 (PTBP1) is an RNA binding protein required for the growth of neurons and may directly transform multiple normal human cells into functioning neurons in vitro and in vivo when expressed at low levels. As a result, we identified it as a key to inhibiting cancer cell proliferation by boosting glioblastoma cell neural differentiation. Methods: Immunocytofluorescence (ICF) targeting TUJ1, MAP2, KI67, and EdU were utilized to evaluate glioblastoma cell reprogramming under PTBP1 knockdown or other conditions. PTBP1 and other target genes were detected using Western blotting and qRT-PCR. Activating protein phosphatase 2A (PP2A) and RhoA were detected using specific kits. CCK8 assays were employed to detect cell viability. Bioluminescence, immunohistofluorescence (IHF), and Kaplan-Meier survival analyses were utilized to demonstrate the in vivo reprogramming efficiency of PTBP1 knockdown in U87 murine glioblastoma model. In this study, RNA-seq technology was used to examine the intrinsic pathway. Results: The expression of TUJ1 and MAP2 neural markers, as well as the absence of KI67 and EdU proliferative markers in U251, U87, and KNS89 cells, indicated that glioblastoma cell reprogramming was successful. In vivo, U87 growth generated xenografts was substantially shrank due to PTBP1 knockdown induced neural differentiation, and these tumor-bearing mice had a prolonged survival time. Following RNA-seq, ten potential downstream genes were eliminated. Lentiviral interference and inhibitors blocking tests demonstrated that UNC5B receptor and its downstream signaling were essential in the neural differentiation process mediated by PTBP1 knockdown in glioblastoma cells. Conclusions: Our results indicate that PTBP1 knockdown promotes neural differentiation of glioblastoma cells via UNC5B receptor, consequently suppressing cancer cell proliferation in vitro and in vivo, providing a promising and feasible approach for glioblastoma treatment.

Transsynaptic cerebellin 4-neogenin 1 signaling mediates LTP in the mouse dentate gyrus.

Five decades ago, long-term potentiation (LTP) of synaptic transmission was discovered at entorhinal cortex→dentate gyrus (EC→DG) synapses, but the molecular determinants of EC→DG LTP remain largely unknown. Here, we show that the presynaptic neurexin­ligand cerebellin-4 (Cbln4) is highly expressed in the entorhinal cortex and essential for LTP at EC→DG synapses, but dispensable for basal synaptic transmission at these synapses. Cbln4, when bound to cell-surface neurexins, forms transcellular complexes by interacting with postsynaptic DCC (deleted in colorectal cancer) or neogenin-1. DCC and neogenin-1 act as netrin and repulsive guidance molecule-a (RGMa) receptors that mediate axon guidance in the developing brain, but their binding to Cbln4 raised the possibility that they might additionally function in the mature brain as postsynaptic receptors for presynaptic neurexin/Cbln4 complexes, and that as such receptors, DCC or neogenin-1 might mediate EC→DG LTP that depends on Cbln4. Indeed, we observed that neogenin-1, but not DCC, is abundantly expressed in dentate gyrus granule cells, and that postsynaptic neogenin-1 deletions in dentate granule cells blocked EC→DG LTP, but again did not affect basal synaptic transmission similar to the presynaptic Cbln4 deletions. Thus, binding of presynaptic Cbln4 to postsynaptic neogenin-1 renders EC→DG synapses competent for LTP, but is not required for establishing these synapses or for otherwise enabling their function.

[Netrin-1, a novel antitumoral target]. / La nétrine-1, une nouvelle cible antitumorale.

Netrin-1, a secreted molecule that was first described for its role in guidance during embryogenesis, was then brought to light for its overexpression in a large number of aggressive cancers. Netrin-1 is a ligand of "dependence receptors". In adults, the interaction between Netrine-1 and these receptors triggers the survival, proliferation, and migration of different cell types. This will confer better survival properties to tumor cells, making them more prone to form aggressive tumors. A recently developed novel therapy aims at inhibiting the binding of Netrin-1 to these receptors in order to trigger cell death by apoptosis. This article presents a review of the functional characteristics of the Netrin-1 molecule, and the potential effects of a novel targeted therapy against Netrin-1 that could lead to very promising results in combination with conventional anti-cancer treatments.

Title: La nétrine-1, une nouvelle cible antitumorale. Abstract: La nétrine-1, une molécule sécrétée mise en évidence pour son rôle de guidage au cours de l'embryogenèse, a été également décrite pour être surexprimée dans de nombreux cancers agressifs. Elle est le ligand de récepteurs dits « à dépendance ¼, à l'origine, chez l'adulte, de la survie, de la prolifération et de la migration de différents types cellulaires, ce qui confère aux cellules cancéreuses des propriétés avantageuses leur permettant de se développer sous forme de tumeurs agressives. Une stratégie thérapeutique consiste à inhiber l'interaction de la nétrine-1 avec son récepteur, ce qui déclenche la mort des cellules par apoptose. Cet article présente une revue des caractéristiques fonctionnelles de cette molécule et les effets potentiels d'une nouvelle thérapie ciblée sur la nétrine-1, dont la combinaison avec les traitements conventionnels pourrait être des plus prometteurs.