Netrin-1 Expression Research Articles

Retracted: Expression and Relationship of Netrin-1, DCC, UNC5B, and VEGF in Villous Tissues of Patients with Delayed Abortion.

[This retracts the article DOI: 10.1155/2022/6283372.].

Netrin-1 controls inflammation in response to ischemic stroke through altering microglia phenotype.

The current approaches that are used to treat ischemic stroke suffer from poor targeting, lack of effectiveness, and potential off-target effects, necessitating the development of new therapeutic strategies to enhance neuronal cell survival and regeneration. This study aimed to investigate the role of microglial Netrin-1 in ischemic stroke, a topic that has not been fully understood. Netrin-1 levels and its primary receptor expressions were investigated in cerebral microglia from acute ischemic stroke patients and age-matched control subjects. A public database (GEO148350), which supplied RNAseq results for rat cerebral microglia in a middle cerebral artery occlusion (MCAO) model, was analyzed to assess the expression of Netrin-1, its major receptors, and genes related to macrophage function. A microglia-specific gene targeting approach and a delivery system allowing for crossing the blood-brain barrier were applied in a mouse model for ischemic stroke to investigate the role of microglial Netrin-1. Netrin-1 receptor signaling in microglia was observed and the effects on microglial phenotype, apoptosis, and migration were analyzed. Across human patients, rat and mouse models, activation of Netrin-1 receptor signaling was mainly conducted via its receptor UNC5a in microglia, which resulted in a shift in microglial phenotype towards an anti-inflammatory or M2-like state, leading to a reduction in apoptosis and migration of microglia. Netrin-1-induced phenotypic change in microglia exerted protective effects on neuronal cells in vivo during ischemic stroke. Our study highlights the potential of targeting Netrin-1 and its receptors as a promising therapeutic strategy for promoting post-ischemic survival and functional recovery.

Sympathetic Innervation Regulates Osteocyte-Mediated Cortical Bone Resorption during Lactation.

Bone undergoes constant remodeling by osteoclast bone resorption coupled with osteoblast bone formation at the bone surface. A third major cell type in the bone is osteocytes, which are embedded in the matrix, are well-connected to the lacunar network, and are believed to act as mechanical sensors. Here, it is reported that sympathetic innervation directly regulates lacunar osteocyte-mediated bone resorption inside cortical bone. It is found that sympathetic activity is elevated in different mouse models of bone loss, including lactation, ovariectomy, and glucocorticoid treatment. Further, during lactation elevated sympathetic outflow induces netrin-1 expression by osteocytes to further promote sympathetic nerve sprouting in the cortical bone endosteum in a feed-forward loop. Depletion of tyrosine hydroxylase-positive (TH+ ) sympathetic nerves ameliorated osteocyte-mediated perilacunar bone resorption in lactating mice. Moreover, norepinephrine activates β-adrenergic receptor 2 (Adrb2) signaling to promote secretion of extracellular vesicles (EVs) containing bone-degrading enzymes for perilacunar bone resorption and inhibit osteoblast differentiation. Importantly, osteocyte-specific deletion of Adrb2 or treatment with a β-blocker results in lower bone resorption in lactating mice. Together, these findings show that the sympathetic nervous system promotes osteocyte-driven bone loss during lactation, likely as an adaptive response to the increased energy and mineral demands of the nursing mother.

Oral ω-3 PUFA supplementation modulates inflammation in adipose tissue depots in morbidly obese women: A randomized trial

ObjectivesObesity is characterized by local and systemic low-grade inflammatory responses. Adipose tissue macrophages (ATM) play decisive roles in inflammation, insulin signaling, and various metabolic dysfunctions. Diets enriched with ω-3 polyunsaturated fatty acids (PUFAs) have been shown to improve health and mitigate pathologic conditions. However, the effects of ω-3 PUFA on adipose tissue inflammation, ATM number, and phenotype are poorly defined in human obesity. The aim of this study was to examine differences in expression of metabolic-inflammatory markers in omental, mesenteric, and subcutaneous fat depots of obese women supplemented with ω-3 PUFAs for 4 wk compared with a low-calorie diet before bariatric surgery. MethodsIn a randomized controlled trial, inflammatory markers in the abdominal adipose tissue and the systemic response in obese women were studied. Patients were treated with a 2-wk low-calorie diet (LCD) or a 4-wk ω-3 PUFA-enriched diet (920 mg eicosapentaenoic acid, 760 mg docosahexaenoic acid daily) before laparoscopic bypass surgery. Omental, mesenteric, and subcutaneous adipose tissue biopsies were collected during surgery and analyzed for quantity and phenotype of ATMs, and profiled for adipokines, cytokines, and signal transduction molecules. ResultsThe chronic inflammatory state characterized by ATM markers was mostly improved by ω-3 PUFAs in visceral adipose tissue. We observed a decreased expression of CD45, CCL2, and CD68, indicating a lower inflammatory state. In patients with type 2 diabetes, ω-3 PUFAs lowered the expression of Netrin-1. ConclusionsCompared with an LCD, a diet enriched with ω-3 PUFAs influences the inflammatory state in different adipose tissue depots, by affecting markers of adipose tissue inflammation, macrophage phenotype, and retention. However, this was not reflected in clinical parameters such as insulin resistance and inflammatory cytokines. Subcutaneous adipose tissue and visceral adipose tissue have different responses to an LCD or a ω-3 PUFA-enriched diet. The presence of diabetes modifies the expression of inflammatory markers.

Netrin-1 mediates nerve innervation and angiogenesis leading to discogenic pain.

Discogenic low back pain (LBP) is associated with nociceptive nerve fibers that grow into degenerated intervertebral discs (IVD) but the etiopathogenesis of disease is not fully understood. The purpose of this study was to clarify the role of Netrin-1 in causing discogenic LBP. The level of nociceptive nerve innervation was examined in disc degenerative patients and rat needle-punctured models by immunohistochemistry. Nucleus pulposus (NP) cells were isolated from IVD tissues of rats and induced degeneration by interleukin-1β (IL-1β) or tumor necrosis factor α (TNFα). The candidate genes related to neuron outgrowth and migration were selected by Next-generation sequencing (NGS). CRISPR/Cas9 was used to knockdown Netrin-1 in NP cells. The impact of Netrin-1 on nerve innervation were evaluated with P2X2、NF200 staining and microfluidics assay. Meanwhile the CD31 staining and transwell assay were used to evaluate the impact of Netrin-1 in angiogenesis. The proteins and RNA extracted from NP cells related to catabolism and anabolism were examined by western blot assay and RT-qPCR experiment. ChIP and luciferase experiments were used to assess the intracellular transcriptional regulation of Netrin-1. Further, a needle-punctured rat model followed by histomorphometry and immunofluorescence histochemistry was used to explore the potential effect of Netrin-1 on LBP in vivo. The level of nerve innervation was increased in severe disc degenerative patients while the expression of Netrin-1 was upregulated. The supernatants of NP cells stimulated with IL-1β or TNFα containing more Netrin-1 could promote axon growth and vascular endothelial cells migration. Knocking down Netrin-1 or overexpressing transcription factor TCF3 as a negative regulator of Netrin-1 attenuated this effect. The needle-punctured rat model brought significant spinal hypersensitivity, nerve innervation and angiogenesis, nevertheless knocking down Netrin-1 effectively prevented disc degeneration-induced adverse impacts. Discogenic LBP was induced by Netrin-1, which mediated nerve innervation and angiogenesis in disc degeneration. Knocking down Netrin-1 by CRISPR/Cas9 or negatively regulating Netrin1 by transcription factor TCF3 could alleviate spinal hypersensitivity. This study on Netrin-1 could provide a new target and theoretical basis for the prevention and treatment for discogenic back pain.

Mechanical Stretch Promotes Neurite Outgrowth of Primary Cultured Dorsal Root Ganglion Neurons via Suppression of Semaphorin 3A-Neuropilin-1/Plexin-A1 Signaling.

Significant attempts have been made to promote neuronal extension and migration in nerve development and regeneration. Although mechanical stretch induces persistent elongation of the axon, the underlying molecular mechanisms are not yet clear. Some axonal guidance cues secreted in the growth cone that affect the axonal growth could attract or repel axons in neurite connection. As semaphorin 3A (Sema3A) is an important repulsion guidance molecule, inhibition of Sema3A has been postulated to promote neuronal development. In this study, the effects of mechanical stretch on dorsal root ganglion neuronal growth and the underlying mechanisms were investigated by assessing the extension direction, neurite length, cell body size, mitochondrial membrane potential, and the expression of Sema3A and its receptors. Our results showed that cell viability significantly increased at tensile strains of 2.5, 5, and 10% for 4 h, with the most prominent effect at 5% tensile strain. Moreover, neurons migrated closer to the stretching direction at 5% tensile strain (0-12 h), while the neurons of the control group moved in a disorderly manner. Furthermore, Sema3A-Neuropilin-1/Plexin-A1 signaling pathway was found to be suppressed after mechanical stretch at 5% tensile strain for 4 h by immunofluorescence staining, immunoprecipitation, and western blot assay. Finally, a Sema3A-SiRNA (SiRNA = small interfering RNA) treatment led to remarkable guidance growth in the stretch-grown neurons. Importantly, there was significant decrease of repulsive cue Sema3A expression and remarkable increase of attractive molecule Netrin-1 expression after mechanical stretching treatment, which jointly promoted neurite outgrowth. This study provides a promising new approach for the development of mechanical stretching therapy or guidance factor-related drugs in injured neuronal regeneration.

Netrin-1 attenuates hepatic steatosis via UNC5b/PPARγ-mediated suppression of inflammation and ER stress

AimsThe current study investigated whether netrin-1 can attenuate hepatic steatosis through PPARγ/autophagy-mediated suppression of inflammation and endoplasmic reticulum (ER) stress in experimental animal models. Main methodsHepatic steatosis was induced by a high-fat diet in experimental mice. Recombinant mouse netrin-1 was administered via the tail vein (1 μg/mouse, once every two days). Serum inflammatory cytokines and hepatic inflammatory and ER stress markers were determined in mice using ELISA and western blotting protocol. Key findingsWe found that netrin-1 expression was significantly increased (P < 0.05) in cultured macrophages treated with supernatants of subcutaneous adipocytes in the presence of palmitate and subcutaneous fat of obese mice. Recombinant netrin-1 treatment promoted PPARγ expression and autophagy, thereby attenuating inflammation and ER stress, lipid accumulation, and the expression of lipogenic proteins in mouse primary hepatocytes. High-fat diet (HFD) treatment increased hepatic inflammation and ER stress, causing hepatic steatosis in experimental mice. However, administration of netrin-1 reversed the effects of HFD on hepatic ER stress and lipid deposition. SignificanceThese results suggest that subcutaneous adipose macrophage-derived netrin-1 ameliorates inflammation and ER stress in the liver, which in turn alleviates hepatic steatosis by enhancing basal PPARγ/autophagy-dependent signaling. The current study sheds light on the pathogenesis of hepatic steatosis in obesity and provides a promising therapeutic approach for treating metabolic-associated fatty liver disease (MAFLD).

Netrin-1 Secreted by Human Osteoarthritic Articular Chondrocytes Promotes Angiogenesis in Vitro.

Netrin-1 expression in articular cartilage is correlated with osteoarthritic changes. We aimed to investigate the contribution of Netrin-1 secreted by human osteoarthritic articular chondrocytes to angiogenesis process in vitro. Human articular chondrocytes were extracted from non-osteoarthritic (n = 10) and osteoarthritic (n = 22) joints obtained from surgical specimens and incubated for 24 hours. Medium conditioned by non-osteoarthritic and osteoarthritic articular chondrocytes were collected. Human umbilical vein endothelial cells (HUVEC) were treated with control and conditioned medium and assessed using assays for cell adherence, migration, and tube formation. Netrin-1 expression and secretion was compared between non-osteoarthritic and osteoarthritic chondrocytes by qPCR, Western blot, and ELISA. The role of chondrocyte-secreted Netrin-1 on HUVEC functions was assessed by immunological neutralization using an anti-Netrin-1 monoclonal antibody. As compared with medium conditioned by non-osteoarthritic chondrocytes, medium conditioned by osteoarthritic chondrocytes permitted tube formation by HUVEC. Both non-osteoarthritic and osteoarthritic chondrocytes expressed Netrin-1 at the RNA and protein levels. At the RNA level, Netrin-1 expression did not differ between non-osteoarthritic and osteoarthritic chondrocytes. At the protein level, Netrin-1 appeared as a full protein of 64 kDa in non-osteoarthritic chondrocytes and as two cleaved proteins of 55 kDa and 64 kDa in osteoarthritic chondrocytes. Immunological neutralization of endogenous Netrin-1 reduced the pro-angiogenic and pro-inflammatory transcriptional profile of HUVEC treated with the medium conditioned by osteoarthritic chondrocytes, as well as their capacities to form tubes. Medium conditioned by osteoarthritic chondrocytes permits tube formation by HUVEC in vitro. This permissive effect is mediated by Netrin-1.

Netrin-1 Stimulates Migration of Neogenin Expressing Aggressive Melanoma Cells.

Netrin-1 is a neural guidance factor that regulates migration and positioning of neural crest-derived cells during embryonic development. Depending on the type of Netrin-1 receptor expression, cells are either attracted or repulsed by Netrin-1. Postnatal expression of Netrin-1 is detected in brain, colon, liver, and kidney, which are common sites of cancer metastasis, including melanoma. Thus, understanding the dynamics between Netrin-1 and its receptors could explain the attraction of melanoma towards these Netrin-1-expressing tissues. Here, we investigate whether the Netrin-1-attractive receptor Neogenin can affect migration of melanoma cells towards a Netrin-1 source. Results from Western blot (WB) analysis show higher expression of Neogenin in aggressive compared to non-aggressive melanoma cells. Cell migration experiments show increased migration of Neogenin-expressing aggressive melanoma cells towards exogenous, soluble recombinant human Netrin-1 and towards a Netrin-1-expressing cell line. Furthermore, WB reveals ERK1/2 activation and increased N-cadherin expression in Neogenin-expressing aggressive melanoma cells treated with rhNetrin-1. Moreover, treatment with anti-Neogenin blocking antibody caused decreased migration towards Netrin-1-expressing cells and reduced ERK1/2 activity in Neogenin-expressing aggressive melanoma cells. These results suggest Neogenin may play a role during migration of melanoma cells towards Netrin-1 via ERK1/2 signaling.

The Relationship Between Serum Netrin-1 Expression Levels and Prognosis in Revascularized Patients with Acute Ischemic Stroke.

This study aimed to explore the relationship between serum netrin-1 expression levels and acute prognosis in patients with acute ischemic stroke (AIS) within 24 hours after revascularization. A total of 121 revascularized patients admitted to the Jinshan Branch of the Shanghai Sixth People's Hospital, China, between July 2019 and July 2021 were selected as study subjects. The primary outcome was the modified Rankin Scale (mRS) score three months after revascularization: patients with an mRS score >2 were classified into the unfavorable prognosis group and others into the favorable prognosis group. Those with serum netrin-1 expression levels greater than the median of all patients were classified into the elevated protein group and others into the decreased protein group. Multivariate logistic regression analysis was used to analyze the independent risk factors for prognosis in patients with AIS after revascularization. The differences between the unfavorable prognosis group and the favorable prognosis group in gender, age, coronary heart disease, and netrin-1 levels were not statistically significant (p > 0.05). However, the National Institute of Health Stroke Scale (NIHSS) scores and number of patients with comorbid hypertension in the unfavorable prognosis group were significantly higher than in the favorable prognosis group (p < 0.05). Multivariate logistic regression analysis showed that NIHSS score before revascularization was an independent risk factor for unfavorable prognosis but that netrin-1 expression levels were not significantly associated with prognosis in patients after revascularization. Serum netrin-1 expression levels in the acute phase are not significantly associated with prognosis in patients with AIS after revascularization.

Implication of Netrin-1 Gain of Expression in Canine Nodal Lymphoma.

Simple SummaryCanine lymphomas represent one of the most frequent groups of neoplasia, for which prognosis may be poor. Treatments are based on polychemotherapy, with variable responses. As in human lymphomas, more and more targeted therapies are studied and developed. Therapy to restore apoptosis in neoplastic cells is one of them. Netrin-1 is a ligand of dependence receptors. When bound to its receptor, a positive signaling is triggered. When unbound, apoptosis is induced. In some human cancers, neoplastic cells can lose the ability to induce apoptosis by overexpressing netrin-1, or by decreasing the receptor expression. We hypothesized a similar pathway in canine lymphomas. We observed increased expression of netrin-1, particularly in high-grade nodal lymphomas. In vitro evaluation of an anti-netrin-1 antibody is encouraging as apoptosis is restored in a T-cell lymphoma cell line. Netrin-1 appears thus as a possible survival factor in dog lymphomas. This study suggests it can be a promising tool for a targeted therapy in lymphoma management in dogs.Netrin-1 is a member of the laminin superfamily, and is known to interact with specific receptors, called dependence receptors. While upon netrin-1 binding these receptors initiate positive signaling, in absence of netrin-1, these receptors trigger apoptosis. Tumor cells can avoid apoptosis by inactivating these receptors or by gaining ligand expression. The aim of the present study was to investigate the expression of netrin-1, the ligand of dependence receptors, in canine healthy lymph nodes (LN), and in lymphomas and to evaluate efficiency of a netrin-1 interfering compound in cell cultures from canine lymphoma. Thirty-two control LN and 169 lymphomas were analyzed through immunohistochemistry. Netrin-1 was expressed in the nucleoli of lymphoid and non-lymphoid cells in controls. Acquisition of a cytoplasmic expression was present in B-cell lymphomas (23.1 % in low-grade and 50.6% in high-grade) and T-cell lymphomas (50.0 % in low-grade and 78.8 % in high-grade), with a significant difference between the high- and low-grade in B-cell lymphomas. Through flow cytometry, we showed a significant increase in netrin-1 expression in either high-grade B-cell and T-cell lymphomas (19 and 5, respectively) compared with healthy LN (5), likewise an RT-qPCR analysis demonstrated a significant increase in netrin-1 expression level in 14 samples of lymphomas compared with eight samples of healthy LN. A T-cell aggressive canine lymphoma cell line and four primary canine nodal lymphomas cell cultures were treated with a netrin-1 interfering antibody. Apoptosis by measuring caspase 3 activity was significantly increased in the cell line and viability was decreased in three of the four primary cell cultures. Together, these data suggest that netrin-1 expression is increased in lymphoma, and more specifically in high-grade lymphomas, and that netrin-1 can act as a survival factor for the neoplastic cells, and so be a therapeutic target.

The Nonproteolytic Intracellular Domain of Membrane-Type 1 Matrix Metalloproteinase Coordinately Modulates Abdominal Aortic Aneurysm and Atherosclerosis in Mice-Brief Report.

MT1-MMP (membrane-type 1 matrix metalloproteinase, MMP-14) is a transmembrane-anchored protein with an extracellular proteinase domain and a cytoplasmic tail devoid of proteolytic functions but capable of mediating intracellular signaling that regulates tissue homeostasis. MT1-MMP extracellular proteolytic activity has been shown to regulate pathological remodeling in aortic aneurysm and atherosclerosis. However, the role of the nonproteolytic intracellular domain of MT1-MMP in vascular remodeling in abdominal aortic aneurysms (AAA) is unknown. We generated a mutant mouse that harbors a point mutation (Y573D) in the MT1-MMP cytoplasmic domain that abrogates the MT1-MMP signaling function without affecting its proteolytic activity. These mice and their control wild-type littermates were subjected to experimental AAA modeled by angiotensin II infusion combined with PCSK9 (proprotein convertase subtilisin/kexin type 9) overexpression and high-cholesterol feeding. The mutant mice developed more severe AAA than the control mice, with concomitant generation of intraaneurysmal atherosclerotic lesions and dramatically increased macrophage infiltration and elastin degradation. Aortic lesion-associated and bone marrow-derived macrophages from the mutant mice exhibited an enhanced inflammatory state and expressed elevated levels of proinflammatory Netrin-1, a protein previously demonstrated to promote both atherosclerosis and AAA. Our findings show that the cytoplasmic domain of MT1-MMP safeguards from AAA and atherosclerotic plaque development through a proteolysis-independent signaling mechanism associated with Netrin-1 expression. This unexpected function of MT1-MMP unveils a novel mechanism of synchronous onset of AAA and atherogenesis and highlights its importance in the control of vascular wall homeostasis.

Exosomal lncRNA PCAT1 Promotes Tumor Circulating Cell-Mediated Colorectal Cancer Liver Metastasis by Regulating the Activity of the miR-329-3p/Netrin-1-CD146 Complex.

Objective This study explored the colorectal cancer exosome lncRNA prostate cancer associated transcript 1– (PCAT1) mediated circulating tumors and the mechanism of cell colorectal cancer liver metastasis. Methods Exosomes were extracted from the primary colorectal cancer (CRC) cell lines HCT116 and SW480 and cultured with T84 and human umbilical vein endothelial (HUVE) cells. The expression of PCAT1 and miR-329-3p was detected by real-time quantitative polymerase chain reaction (RT-qPCR), the expression of Netrin-1, CD146, and epithelial mesenchymal transition (EMT) related proteins was detected by Western blot, the proliferation activity of T84 cells was detected by cell counting kit 8 (CCK-8), and cell migration was detected by Transwell. The expression of the F-actin signal was detected by immunofluorescence after coculture of exosomes with human umbilical vein endothelial cells (HUVECs). Changes in subcutaneous tumor and liver nodule size after PCAT1 deletion were observed in a mouse model of liver metastasis from rectal cancer. Results PCAT1 expression was upregulated in primary cell lines and their exosomes. After exosomes were cocultured with colorectal cancer tumor circulating T84 cells, the expression of Netrin-1 and CD146 was upregulated, the expression of miR-329-3p was downregulated, the proliferation and migration ability of T84 cells were enhanced, and EMT occurred. After knocking down PCAT1, the above phenomenon was reversed. Similarly, after exosomes were cocultured with HUVECs, the expression of the F-actin signal increased, and after PCAT1 was knocked down, the F-actin signal also decreased. PCAT1 regulates miR-329-3p/Netrin-1 and affects the biological behavior of T84 and F-actin signal expression in HUVECs. In a mouse model of colorectal cancer liver metastasis, knocking down PCAT1 significantly reduced the nodules formed by liver metastasis in mice. Conclusions LncRNA PCAT1 derived from colorectal cancer exosomes regulates the activity of the Netrin-1-CD146 complex in circulating tumor cells (CTCs) to promote the occurrence of colorectal cancer EMT and liver metastasis and provides new molecular targets for the treatment of colorectal cancer liver metastasis.

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

BackgroundB-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.MethodsThe 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.ResultsNetrin-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.ConclusionTaken 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.-oqtjpF4fn996fXAD1ptLBVideo

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.

Effect of Netrin-1 on VEGFA Expression in T-ALL Cells and Its Related Mechanism

ELISA assays were applied to detect the levels of Netrin-1 and VEGFA in the bone marrow (BM) samples from children in the T-ALL and control group. The level of Netrin-1 and VEGFA were compared between control children and patients, and the liner correlation between Netrin-1 and VEGFA was analyzed. The T-ALL cells Jurkat and Molt-4 were culture in vitro, and the cells were treated with different concentration of Netrin-1 (0, 25, 50, 100 ng/ml) for 24 h, quantitative RT-PCR (qRT-PCR) and Western blot were used to detect the VEGFA expression in Jurkat, Molt-4 cells. The expression of Netrin-1 receptors in T-ALL cells was detected by qRT-PCR and the interaction between Netrin-1 and receptor in each cells was detected by co-IP. Furthermore, Western blot was used to detect the phosphorylation level of key prateins of AKT signal transduction pathway including Akt and mTOR in T-ALL cells treated with Netrin-1 (100 ng/ml). The expression of VEGFA and phosphorylation of AKT pathway transducers were detected by Western blot, after T-ALL cells treated with Netrin-1 (100 ng/ml) combined with inhibitors specific to Akt or mTOR. The expression level of Netrin-1 and VEGFA in T-ALL patients BM samples were both signi-ficantly higher than that of control group. And the expression level of Netrin-1 was positively correlated with that of VEGFA（r2=0974). With the increase of Netrin-1 concentration, the expression level of VEGFA also increased(P<0.05）. Netrin-1 interacted with its receptor, integrin-β4 at the Netrin-1 concentration of 100 ng/ml. Further, the treatment of Netrin-1 could increase the phosphorylation of Akt and mTOR, which were the key transducers of AKT pathway. After treatment of T-ALL cells with Netrin-1 (100 ng/mL) and Akt inhibitor, the expression of VEGFA and phosphorylation of Akt or mTOR decreased. When the cells were treated with Netrin-1(100 ng/ml) and mTOR inbititor, the phosphorylation level of mTOR and the expression of VEGFA decreased, the phosphorylation level of Akt increased. The expression of Netrin-1 and VEGFA in bone marrow of childred with T-ALL were abnormal, and there was a linear relationship between them. Netrin-1 can interact with its receptor, integrin-β4 and activate AKT transduction pathway to elevate the expression of VEGFA in T-ALL cells.

Bone Marrow Mesenchymal Stem Cells (BMSCs) Expressing Netrin-1 Alleviates Spinal Cord Injury (SCI)-Induced Inflammation

Spinal cord injury (SCI) is a common central nervous system (CNS) injury. Bone marrow mesenchymal stem cells (BMSCs) transplantation is a potential treatment for traumatic SCI. However, the role and mechanism of BMSCs with high expression of Netrin-1 on the repair and inflammation of spinal cord injury cells remains unclear. Our study intends to assess the effect of BMSCs with high Netrin-1 level on the repair of SCI cells. BMSCs or Netrin-1 transfected BMSCs were co-cultured with mechanically injured nerve cells followed by analysis of the differentiation of BMSCs by light microscope, apoptosis activity, expression of TLR-4 and NF-κB, and the TNF-α and IL-1β content in cell supernatant by ELISA. BMSCs with high Netrin-1 expression promoted the proliferation of BMSCs, inhibited apoptosis, and promoted the differentiation of nerve cells along with increased ALK activity, and the expression of GFAP and BDNF. Co-culture with BMSCs or BMSCs with high Netrin-1 expression increased mechanically damaged nerve cell proliferation, decreased apoptosis, downregulated TLR-4 and NF-κB (P &lt; 0.05) with more significant changes after co-culture with BMSCs with high Netrin-1 expression. In conclusion, Netrin-1 can promote BMSCs proliferation and differentiation, and inhibit apoptosis. By inhibiting inflammation, it can promote damaged nerve cell proliferation and repair.

Detection of netrin-1 as a novel biomarker for diagnosis and chemotherapeutic monitoring of lung cancer.

ObjectiveLung cancer has high morbidity and mortality. We aimed to determine the value of netrin-1 for the diagnosis and chemotherapeutic monitoring of lung cancer.MethodsThirty pairs of lung cancer tissues and serum were collected. Netrin-1 expression was detected by immunohistochemistry and enzyme-linked immunosorbent assay. Netrin-1 expression was downregulated in A549 cells using small interfering RNA, and the effect of netrin-1 on cisplatin-induced lung cancer cell apoptosis was determined by flow cytometry.ResultsNetrin-1-positivity was significantly higher in lung cancer tissues than in paracarcinoma tissues and high expression of netrin-1 was closely related to a poor prognosis. Serum netrin-1 levels were also significantly higher in lung cancer patients than in healthy donors, and were higher in patients with lung cancer before the beginning of chemotherapy compared with after the completion of four cycles of chemotherapy. Netrin-1 knockdown increased the rate of cisplatin-induced apoptosis in A549 cells.ConclusionsNetrin-1 expression was increased in tissues and serum from lung cancer patients and decreased after chemotherapy, suggesting that it may be a potential diagnostic marker and indicator of chemosensitivity. Netrin-1 may participate in cisplatin resistance by reducing apoptosis, thus providing a new strategy for addressing chemoresistance in patients with lung cancer.

Inactivation of axon guidance molecule netrin-1 in human colorectal cancer by an epigenetic mechanism

Netrin-1, the protein product of the NTN1 gene, is an axon guidance molecule implicated in regulation of cell survival and tumorigenesis. Expression of the netrin-1 receptors deleted in colorectal cancer (DCC) and uncoordinated 5 homolog (UNC5H) is frequently silenced in colorectal cancer (CRC) by either loss of heterozygosity or epigenetic mechanisms. However, netrin-1 expression and regulation in CRC are mostly unknown. Here, we report that NTN1 expression is significantly reduced in most CRC tissues compared to the adjacent normal intestinal mucosa, and that NTN1 DNA methylation is significantly higher in CRCs (24.6%) than in the adjacent normal intestinal mucosa (4.0%). In 6 CRC cell lines, NTN1 expression is low. Treatment with 5-Aza-2′-deoxycytidine increased expression of NTN1 in CRC cell lines, indicating that DNA methylation represses NTN1 transcription in CRCs. NTN1 DNA hypermethylation was significantly associated with advanced CRC disease. Median netrin-1 serum levels were significantly decreased in CRC patients (330.1 pg/mL) compared with normal individuals (438.6 pg/mL). Our results suggest that netrin-1 is a candidate biomarker for CRC.

Suppression of axonal attractant netrin-1 injured dopaminergic neuronal and motor function of mice during manganese overexposure.

Manganese (Mn) can accumulate in the striatum through the blood-brain barrier and cause neurotoxicity. It is mainly due to the decrease of dopamine (DA) levels in the striatum, which leads to extrapyramidal dysfunction. Netrin-1, as an axon guidance factor, can regulate the normal transmission of DA. However, few people have explored the role of netrin-1 in Mn-induced neurotoxicity. The purpose of the present study is to verify whether overexposure of Mn inhibits the axon attractant netrin-1, thereby damaging dopaminergic neuronal and motor function of mice. Here, we found that excessive Mn exposure reduces the expression of striatum netrin-1, tyrosine hydroxylase, DA receptor D3, and dopamine transporter1, and the levels of serum netrin-1, and promotes dopaminergic neuronal and striatum injury, leading to DA transmission and motor dysfunction. Notably, recombinant mouse netrin-1 protein significantly antagonized Mn-induced neurotoxicity. These findings suggest that netrin-1 participates in Mn-induced motor dysfunction. Our findings may provide an experimental basis for fully elucidating the effects of Mn-induced neurotoxicity.