Netrin-1-CD146 and netrin-1-S100A9 are associated with early stage of lymph node metastasis in colorectal cancer.

BACKGROUND: The netrin-1/CD146 pathway regulates colorectal cancer (CRC) liver metastasis, angiogenesis, and vascular development. However, few investigations have yet examined the biological function of netrin-1/CD146 complex in CRC. In this work, we investigated the relationship between the netrin-1/CD146 axis and S100 proteins in sentinel lymph node, and revealed a possible new clue for vascular metastasis of CRC. METHODS: The expression levels of netrin-1 and CD146 proteins in CRC, as well as S100A8 and S100A9 proteins in the sentinel lymph nodes were determined by immunohistochemistry. Using GEPIA and UALCAN, we analyzed netrin-1 and CD146 gene expression in CRC, their association with CRC stage, and their expression levels and prognosis in CRC patients. RESULTS: The expression level of netrin-1 in N1a+1b (CRC lymphatic metastasis groups, exculded N1c) was positively increased with N0 (p = 0.012). The level of netrin-1 protein was positively correlated with CD146 protein (p < 0.05). The level of S100A9 protein was positively correlated with CD146 protein (r = 0.492, p = 0.007). Moreover, netrin-1 expression was obviously correlated with S100A9 expression in the N1 stage (r = 0.867, p = 0.000). CD146 level was correlated with S100A9 level in the N2 stage (r = 0.731, p = 0.039). CD146 mRNA expression was higher in normal colorectal tissues than in CRC (p < 0.05). Netrin-1 and CD146 expression were not significantly associated with the tumor stages and prognosis of patients with CRC (p > 0.05). CONCLUSIONS: The netrin-1/CD146 and netrin-1/S100A9 axis in CRC tissues might related with early stage of lymph node metastasis, thus providing potential novel channels for blocking lymphatic metastasis and guiding biomarker discovery in CRC patients.

Netrin 1 as a biomarker in cancer: scoping diagnostic, prognostic, and therapeutic perspectives with a focus on oral squamous cell carcinoma.

Goal of the review: The utilization of biomarkers to predict cancer risk, prognosis, and treatment outcomes is paramount. Netrin-1 (NTN1), known for its role in commissural axon guidance during embryonic development, has emerged as a versatile molecule with significant implications in cancer and neurobiology. Structurally resembling laminin, Netrin-1 regulates neuronal connectivity and plasticity in adulthood, influencing axonal and dendritic growth, neurotransmission, and cell migration. In addition to its neurological functions, Netrin-1 is increasingly recognized for its involvement in maintaining epithelial tissue and its regulatory roles in fundamental cellular processes, including adhesion, proliferation, differentiation, apoptosis, and angiogenesis. In cancer biology, Netrin-1's interactions with its receptors, such as DCC [Deleted in Colorectal Cancer] and UNC5 (a homolog of DCC), have been implicated in tumor progression across various physiological systems. Elevated levels of Netrin-1 in colorectal cancer and head and neck squamous cell carcinoma are correlated with increased tumorigenic potential, mediated through pathways involving NFκB activation and anti-apoptotic mechanisms. Mechanically induced hypermethylation and downstream signaling cascades that inhibit apoptosis and promote cell survival are observed upon Netrin-1 binding to DCC. Furthermore, Netrin-1 shows promise as a biomarker for detecting inflammatory activity in diseases such as multiple sclerosis and as a potential diagnostic, prognostic, and therapeutic indicator in oral squamous cell carcinoma. Elevated levels of Netrin-1 in bodily fluids, alongside immunohistochemical evidence, support its potential as a valuable clinical marker in cancer management. This abstract emphasizes Netrin-1's diverse biological roles, underscoring its potential as a diagnostic tool and therapeutic target in cancer research. The need for further exploration of Netrin-1's molecular interactions and clinical applications is urgent and crucial to advance personalized medicine approaches and enhance patient outcomes in oncology and neurology.

Anti-Netrin-1 decorated nanoparticles combined with chemotherapy for the treatment of triple-negative breast cancer.

Nanoparticle's success as drug delivery systems for cancer treatment has been achieved through passive targeting mechanisms. However, tumor heterogeneity and rapid drug clearance limit the treatment efficacy. Improved outcomes and selective drug release can be achieved by grafting ligands at the surface of nanocarriers that bind molecules overexpressed in the tumor microenvironment (TME). In this work, we developed a docetaxel-loaded nanoemulsions (NEs) binding an anti-netrin-1 monoclonal antibody (NP137) to selectively target the netrin-1 protein overexpressed in many different tumors. The goal is to refine a combined approach utilizing NP137 and docetaxel as an improved tumor-targeting chemotherapeutic agent for addressing triple-negative breast cancer (TNBC). Several factors have been considered for the optimization of the active targeted drug delivery system via the click-chemistry conjugation, as the impact of PEGylated surfactant that stabilize the NEs shell on conjugation efficiency, cytocompatibility with EMT6 cell line and colloidal stability over time of NEs. Results showed that a 660 Da PEG chain length contributed to NEs colloidal stability and had no impact on cell viability or on the antibody binding ability for its ligand after surface conjugation. Moreover, docetaxel was encapsulated into the oily core of NEs, with an encapsulation efficiency of 70 %. To validate our treatment strategy in vivo, the 4T1 murine breast cancer model was used. As a result, the comparison of active-targeted and non-targeted NEs revealed that only active-targeted NE could decrease the tumor growth rate.

Insights from the neural guidance factor Netrin-1 into neurodegeneration and other diseases.

Netrin-1 was initially discovered as a neuronal growth cue for axonal guidance, and its functions have later been identified in inflammation, tumorigenesis, neurodegeneration, and other disorders. We have recently found its alterations in the brains with Alzheimer's disease, which might provide important clues to the mechanisms of some unique pathologies. To provide better understanding of this promising molecule, we here summarize research progresses in genetics, pathology, biochemistry, cell biology and other studies of Netrin-1 about its mechanistic roles and biomarker potentials with an emphasis on clinical neurodegenerative disorders in order to expand understanding of this promising molecular player in human diseases.

Expression of netrin-1 in uterine serous carcinoma and its association with prognosis.

BACKGROUND/OBJECTIVES: At present, there are few biomarkers used to predict the prognosis of uterine serous carcinoma (USC). Netrin-1 may be a promising biomarker candidate. We investigated netrin-1 expression in USC tissues and healthy endometrial tissues to determine its relevance to disease prognosis. MATERIALS AND METHODS: Netrin-1 expression was examined in the tissues of 48 patients with USC and 30 patients with healthy benign endometrial tissues via immunohistochemistry. RESULTS: None of the healthy tissues were stained with netrin-1. In tumor tissues, the overall positivity rate of netrin-1 was 75%, detected as high expression in 17 patients (35%) and low in 19 (40%). Patients who had tumors with no netrin-1 expression (n = 12) had a median overall survival (OS) of 60.0 months (95% confidence interval [CI], 47-98), whereas patients who had tumors with low to strong netrin-1 expression (n = 33) had a lower median OS of 50 months, but the difference was not statistically significant (95% CI, 58-108; P = 0.531). Disease-free survival (DFS) was not statistically significant between the groups (95% CI, 67.7-115.9; P = 0.566). Patients with a tumor diameter ≥2 cm had higher netrin-1 expression than those with a tumor diameter of 2 cm (P = 0.027). We did not find any difference in overall and DFS when age, tumor stage, histology, tumor diameter, p53 status, lymphovascular space invasion, myometrial invasion, and lymph node metastasis were compared according to netrin-1 expression (P > 0.05). CONCLUSION: Netrin-1 was expressed in USC but not in healthy tissues. Its expression was not associated with OS or DFS.

Netrin-1 as A neural guidance protein in development and reinnervation of the larynx.

Neural guidance proteins participate in motor neuron migration, axonal projection, and muscle fiber innervation during development. One of the guidance proteins that participates in axonal pathfinding is Netrin-1. Despite the well-known role of Netrin-1 in embryogenesis of central nervous tissue, it is still unclear how the expression of this guidance protein contributes to primary innervation of the periphery, as well as reinnervation. This is especially true in the larynx where Netrin-1 is upregulated within the intrinsic laryngeal muscles after nerve injury and where blocking of Netrin-1 alters the pattern of reinnervation of the intrinsic laryngeal muscles. Despite this consistent finding, it is unknown how Netrin-1 expression contributes to guidance of the axons towards the larynx. Improved knowledge of Netrin-1's role in nerve regeneration and reinnervation post-injury in comparison to its role in primary innervation during embryological development, may provide insights in the search for therapeutics to treat nerve injury. This paper reviews the known functions of Netrin-1 during the formation of the central nervous system and during cranial nerve primary innervation. It also describes the role of Netrin-1 in the formation of the larynx and during recurrent laryngeal reinnervation following nerve injury in the adult.

Netrin-1 Role in Nociceptive Neuron Sprouting through Activation of DCC Signaling in a Rat Model of Bone Cancer Pain.

BACKGROUND: Bone cancer pain (BCP) is a common primary or metastatic bone cancer complication. Netrin-1 plays an essential role in neurite elongation and pain sensitization. This study aimed to determine the role of netrin-1 from the metastatic bone microenvironment in BCP development and identify the associated signaling pathway for the strategy of BCP management. METHODS: The rat BCP model was established by intratibial implantation of Walker 256 cells. Von Frey filaments measured the mechanical pain threshold. Movement-induced pain was assessed using limb use scores. Expressions of associated molecules in the affected tibias or dorsal root ganglia (DRG) were measured by immunofluorescence, immunohistochemistry, real-time quantitative polymerase chain reaction, or western blotting. Transduction of deleted in colorectal cancer (DCC) signaling was inhibited by intrathecal injection of DCC-siRNA. RESULTS: In BCP rats, the presence of calcitonin gene-related peptide (CGRP)-positive nerve fibers increased in the metastatic bone lesions. The metastatic site showed enrichment of well-differentiated osteoclasts and expressions of netrin-1 and its attractive receptor DCC. Upregulation of DCC and increased phosphorylation levels of focal adhesion kinase (FAK) and Rac family small GTPase 1/Cell division cycle 42 (Rac1/Cdc42) were found in the DRG. Intrathecal administration of DCC-siRNA led to a significant reduction in FAK and Rac1/Cdc42 phosphorylation levels in the DRG, decreased nociceptive nerve innervation, and improved pain behaviors. CONCLUSIONS: Netrin-1 may contribute to the activation of the BCP by inducing nociceptive nerve innervation and improving pain behaviors.

Adhesion-clutch between DCC and netrin-1 mediates netrin-1-induced axonal haptotaxis.

The growth cone, a motile structure located at the tip of growing axons, senses extracellular guidance cues and translates them into directional forces that drive axon outgrowth and guidance. Axon guidance directed by chemical cues on the extracellular adhesive substrate is termed haptotaxis. Recent studies reported that netrin-1 on the substrate functions as a haptotactic axon guidance cue. However, the mechanism mediating netrin-1-induced axonal haptotaxis remains unclear. Here, we demonstrate that substrate-bound netrin-1 induces axonal haptotaxis by facilitating physical interactions between the netrin-1 receptor, DCC, and the adhesive substrates. DCC serves as an adhesion receptor for netrin-1. The clutch-linker molecule shootin1a interacted with DCC, linking it to actin filament retrograde flow at the growth cone. Speckle imaging analyses showed that DCC underwent either grip (stop) or retrograde slip on the adhesive substrate. The grip state was more prevalent on netrin-1-coated substrate compared to the control substrate polylysine, thereby transmitting larger traction force on the netrin-1-coated substrate. Furthermore, disruption of the linkage between actin filament retrograde flow and DCC by shootin1 knockout impaired netrin-1-induced axonal haptotaxis. These results suggest that the directional force for netrin-1-induced haptotaxis is exerted on the substrates through the adhesion-clutch between DCC and netrin-1 which occurs asymmetrically within the growth cone.

Netrin-1 feedforward mechanism promotes pancreatic cancer liver metastasis via hepatic stellate cell activation, retinoid, and ELF3 signaling.

The biology of metastatic pancreatic ductal adenocarcinoma (PDAC) is distinct from that of the primary tumor due to changes in cell plasticity governed by a distinct transcriptome. Therapeutic strategies that target this distinct biology are needed. We detect an upregulation of the neuronal axon guidance molecule Netrin-1 in PDAC liver metastases that signals through its dependence receptor (DR), uncoordinated-5b (Unc5b), to facilitate metastasis in vitro and in vivo. The mechanism of Netrin-1 induction involves a feedforward loop whereby Netrin-1 on the surface of PDAC-secreted extracellular vesicles prepares the metastatic niche by inducing hepatic stellate cell activation and retinoic acid secretion that in turn upregulates Netrin-1 in disseminated tumor cells via RAR/RXR and Elf3 signaling. While this mechanism promotes PDAC liver metastasis, it also identifies a therapeutic vulnerability, as it can be targeted using anti-Netrin-1 therapy to inhibit metastasis using the Unc5b DR cell death mechanism.

Enhanced Neuron Growth and Electrical Activity by a Supramolecular Netrin-1 Mimetic Nanofiber.

Neurotrophic factors are essential not only for guiding the organization of the developing nervous system but also for supporting the survival and growth of neurons after traumatic injury. In the central nervous system (CNS), inhibitory factors and the formation of a glial scar after injury hinder the functional recovery of neurons, requiring exogenous therapies to promote regeneration. Netrin-1, a neurotrophic factor, can initiate axon guidance, outgrowth, and branching, as well as synaptogenesis, through activation of deleted in colorectal cancer (DCC) receptors. We report here the development of a nanofiber-shaped supramolecular mimetic of netrin-1 with monomers that incorporate a cyclic peptide sequence as the bioactive component. The mimetic structure was found to activate the DCC receptor in primary cortical neurons using low molar ratios of the bioactive comonomer. The supramolecular nanofibers enhanced neurite outgrowth and upregulated maturation as well as pre- and postsynaptic markers over time, resulting in differences in electrical activity similar to neurons treated with the recombinant netrin-1 protein. The results suggest the possibility of using the supramolecular structure as a therapeutic to promote regenerative bioactivity in CNS injuries.

Netrina-1 en sangre como biomarcador para la detección del cáncer de endometrio / Serum Netrin-1 as a biomarker for Endometrium Cancer detection

RESUMEN Investigar la relación de la netrina-1 preoperatoria con factores clinicopatológicos y pronósticos importantes y los niveles de corte adecuados en pacientes con cáncer de endometrio. En este estudio prospectivo y observacional, el grupo de casos y el de controles se seleccionaron entre las pacientes que acudieron a la Clínica de Oncología Ginecológica. Se extrajeron 4 mL de sangre venosa en un tubo de bioquímica de cada paciente durante el período preoperatorio. Los valores de netrina para predecir la presencia de malignidad se analizaron mediante el análisis de la curva ROC (receiver operating characteristics). El valor de corte se calculó según el índice de Youden. En el estudio, el valor de corte de malignidad según el nivel de netrina fue determinado en 645,50 mg/dL en el análisis ROC (utilizando el índice de Youden). La probabilidad de malignidad en individuos con valores de netrina superiores a este punto de corte fue del 78,2% (IC 95%: 0,680 a 0,884). La sensibilidad de la netrina para mostrar la probabilidad de malignidad en este valor de corte fue del 87,5% y la especificidad del 63,6%. La netrina-1 puede ser un biomarcador potencial para la detección del cáncer de endometrio y la evaluación de su pronóstico.

ABSTRACT To investigate the relationship of preoperative netrin-1 with important clinicopathological and prognostic factors and appropriate cut-off levels in patients with endometrial cancer. In this prospective, observational study, the case and control group were selected among patients who applied to the Gynecological Oncology Clinic. Four mL of venous blood was drawn into a biochemistry tube from each patient during the preoperative period. Netrin values in predicting the presence of malignity were analyzed using ROC (receiver operating characteristics) curve analysis. The cut-off value was calculated according to the Youden index. In the study, the cut-off value of malignancy according to the netrin level was determined as 645.50 mg/dL in the ROC analysis (using the Youden index). The probability of malignancy in individuals with Netrin values above this cut-off was 78.2% (95% CI 0.680-0.884). The sensitivity of netrin in showing the probability of malignancy at this cut-off value was 87.5%, and the specificity 63.6%. Netrin-1 can be a potential biomarker for endometrial cancer detection and prognosis evaluation.

Activation of vascular endothelial cells by synovial fibrosis promotes Netrin-1-induced sensory nerve sprouting and exacerbates pain sensitivity.

Synovial fibrosis is one of the most dominant histopathological changes in osteoarthritis of the knee (KOA), and activation of vascular endothelial cells in synovial fibrosis is both an important factor in mediating pain in KOA and a major contributor to the generation of pain signals. At the same time, angiogenesis and nerve fibres are more likely to underlie the pathology of pain induced by synovial fibrosis. In the present study, we established a co-culture model of human umbilical vein endothelial cells (HUVECs) with dorsal root ganglion (DRG) and detected tissue and cellular Netrin-1, vascular cell adhesion molecule-1 (VCAM-1), intercellular cell adhesion molecule-1 (ICAM-1), growth-associated protein-43 (GAP43), colorectal cancer deleted (DCC), uncoordinated 5 (UNC5), and the related expression of calcitonin gene-related peptide (CGRP), substance P (SP) and nerve growth factor (NGF) in supernatant by ELISA to investigate the intervention of vascular endothelial cell activation on sensory nerve sprouting exacerbating peripheral pain sensitivity and to investigate the effect of Netrin-1 from the perspective of Netrin-1 secretion to illustrate its effector mechanism.

Exosomes derived from human umbilical cord mesenchymal stem cells loaded with RVG-Lamp2b and Netrin-1 promotes Schwann cell invasion and migration.

BACKGROUND: Netrin-1 has a neuroprotective effect by regulating angiogenesis, autophagy, apoptosis, and neuroinflammation. This study investigated the effects of netrin-1 delivery to mouse Schwann cells and vascular endothelial cells using exosomes modified with rabies virus glycoprotein (RVG) peptides. MATERIALS AND METHODS: RVG-Lamp2b and/or Netrin-1 were overexpressed in human umbilical cord mesenchymal stem cells to obtain exosomes modified with RVG-Lamp2b and/or loaded with Netrin-1. Then, exosomes were labeled with carboxyfluorescein diacetate succinimidyl ester and co-cultured with mouse Schwann cells and endothelial cells. Netrin-1 expression in Schwann cells and endothelial cells was measured using quantitative polymerase chain reaction and immunoblotting. Moreover, methyl thiazolyl tetrazolium assays and Transwell assays were used to detect proliferation, migration, and invasion of Schwann cells and endothelial cells. RESULTS: Exosomes with RVG-Lamp2b entered Schwann cells more readily compared with the exosomes without RVG-Lamp2b. Meanwhile, this was not the case in endothelial cells. Netrin-1-loaded exosomes significantly promoted Netrin-1 expression, cell proliferation, migration, invasion, and epithelial-mesenchymal transition in Schwann cells and endothelial cells. These effects were further enhanced by Netrin-1-loaded exosomes modified with RVG-Lamp2b in Schwann cells, but not in endothelial cells. CONCLUSION: HucMSC-derived exosomes loaded with RVG-Lamp2b and Netrin-1 promote proliferation, migration, and invasion of Schwann cells.

Differential expression of PLAC1 and Netrin-1 in liver metastasis of colorectal cancer and its predictive value.

OBJECTIVE: To explore the differential expression of placental specific gene 1 (PLAC1) and neurite guidance factor 1 (netrin-1) in colorectal cancer (CRC) liver metastasis and its predictive value. METHODS: Paraffin specimens of primary CRC were selected, including 60 simple colorectal cancer specimens and 47 liver metastasis specimens. At the same time, 40 cases of normal colorectal mucosa were taken as the control group. The expression of PLAC1 and Netrin-1 in tissue was detected by immunohistochemistry (IHC). The correlation between PLAC1 and Netrin-1 expression and clinicopathological characteristics of patients with CRC liver metastases was analyzed. Logistic analysis was adopted to analyze the influencing factors of liver metastasis in CRC. A prediction model was established and ROC curve was used to detect the discrimination of the prediction model. The clinical value of PLAC1 and netrin-1 in predicting liver metastasis of CRC was analyzed using ROC curve. The relationship between the expression of PLAC1 and netrin-1 and the prognosis of CRC patients with liver metastasis was analyzed using Kaplan Meier survival curve. RESULTS: The positive staining of PLAC1 and netrin-1 was mainly located in the cytoplasm by IHC detection. Positive expression of PLAC1 and netrin-1 in CRC tissues was markedly higher than that in normal colorectal mucosal epithelium (P < 0.05). Positive expression of PLAC1 in metastatic group was higher than that in non-metastatic group without significant difference (P > 0.05). The metastasis group had much higher positive expression of netrin-1 than the non-metastasis group (P < 0.05). The content of PLAC1 in the tissues of CRC with liver metastasis had a close relationship with differentiation degree and lymph node metastasis (P < 0.05). The expression of Netrin-1 in the tissues of CRC with liver metastasis was associated with Dukes stage, differentiation degree and lymph node metastasis (P < 0.05). Logistic regression analysis showed that Dukes stage, differentiation, lymph node metastasis, CEA, Alb and D-dimer were the independent risk factors for liver metastasis of CRC (P < 0.05). The model was constructed according to the regression coefficients and constant terms, and the discrimination of the prediction model was evaluated using ROC curve, with the AUC of 0.903 (95% CI 0.831 ~ 0.975), the sensitivity of 93.80%, the specificity of 80.00%, and the Jordan index of 0.738. The AUC of PLAC1 and netrin-1 alone and combined detection to predict liver metastasis of CRC were 0.805, 0.793 and 0.921, respectively. The survival time of patients with positive PLAC1 and netrin-1 expression were sharply shorter than that of the patients with negative expression (P < 0.05). CONCLUSIONS: The expression of PLAC1 and netrin-1 was strongly increased in CRC with liver metastasis, which had a certain clinical value in predicting liver metastasis of CRC. Dukes stage, differentiation degree, lymph node metastasis, CEA, Alb and D-dimer were independent risk factors for liver metastasis of CRC, and the model based on these indicators had good discrimination for effectively evaluating the risk of liver metastasis in CRC.

Human Retinal Ganglion Cells Respond to Evolutionarily Conserved Chemotropic Cues for Intra Retinal Guidance and Regeneration.

Retinal ganglion cells (RGCs) connect the retina with the higher centers in the brain for visual perception. Their degeneration leads to irreversible vision loss in patients with glaucoma. The mechanism underlying human RGCs (hRGCs) axon growth and guidance remains poorly understood because hRGCs are born during development and connections with the central targets are established before birth. Here, using RGCs directly generated from human embryonic stem cells, we demonstrate that hRGCs express a battery of guidance receptors. These receptors allow hRGCs to read the spatially arrayed chemotropic cues in the developing rat retina for the centripetal orientation of axons toward the optic disc, suggesting that the mechanism of intraretinal guidance is conserved in hRGCs. The centripetal orientation of hRGCs axons is not only in response to chemorepulsion but also involves chemoattraction, mediated by Netrin-1/DCC interaction. The spatially arrayed chemotropic cues differentially influence hRGCs physiological responses, suggesting that neural activity of hRGCs and axon growth may be coupled during inter-retinal guidance. In addition, we demonstrate that Netrin-1/DCC interaction, besides promoting axon growth, facilitates hRGCs axon regeneration by recruiting the mTOR signaling pathway. The diverse influence of Netrin-1/DCC interaction ranging from axon growth to regeneration may involve recruitment of multiple intracellular signaling pathways as revealed by transcriptome analysis of hRGCs. From the perspective of ex vivo stem cell approach to glaucomatous degeneration, our findings posit that ex vivo generated hRGCs can read the intraretinal cues for guidance toward the optic disc, the first step required for connecting with the central target to restore vision.

Gαi1/3 mediate Netrin-1-CD146-activated signaling and angiogenesis.

Netrin-1 binds to the high-affinity receptor CD146 to activate downstream signaling and angiogenesis. Here, we examine the role and underlying mechanisms of G protein subunit alpha i1 (Gαi1) and Gαi3 in Netrin-1-induced signaling and pro-angiogenic activity. In mouse embryonic fibroblasts (MEFs) and endothelial cells, Netrin-1-induced Akt-mTOR (mammalian target of rapamycin) and Erk activation was largely inhibited by silencing or knockout of Gαi1/3, whereas signaling was augmented following Gαi1/3 overexpression. Netrin-1 induced Gαi1/3 association with CD146, required for CD146 internalization, Gab1 (Grb2 associated binding protein 1) recruitment and downstream Akt-mTOR and Erk activation. Netrin-1-induced signaling was inhibited by CD146 silencing, Gab1 knockout, or Gαi1/3 dominant negative mutants. Netrin-1-induced human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation were inhibited by Gαi1/3 short hairpin RNA (shRNA), but were potentiated by ectopic Gαi1/3 overexpression. In vivo, intravitreous injection of Netrin-1 shRNA adeno-associated virus (AAV) significantly inhibited Akt-mTOR and Erk activation in murine retinal tissues and reduced retinal angiogenesis. Endothelial knockdown of Gαi1/3 significantly inhibited Netrin1-induced signaling and retinal angiogenesis in mice. Netrin-1 mRNA and protein expression were significantly elevated in retinal tissues of diabetic retinopathy (DR) mice. Importantly, silence of Netrin-1, by intravitreous Netrin-1 shRNA AAV injection, inhibited Akt-Erk activation, pathological retinal angiogenesis and retinal ganglion cells degeneration in DR mice. Lastly, Netrin-1 and CD146 expression is significantly increased in the proliferative retinal tissues of human proliferative diabetic retinopathy patients. Together, Netrin-1 induces CD146-Gαi1/3-Gab1 complex formation to mediate downstream Akt-mTOR and Erk activation, important for angiogenesis in vitro and in vivo.

A tug of war between DCC and ROBO1 signaling during commissural axon guidance.

Dynamic and coordinated axonal responses to changing environments are critical for establishing neural connections. As commissural axons migrate across the CNS midline, they are suggested to switch from being attracted to being repelled in order to approach and to subsequently leave the midline. A molecular mechanism that is hypothesized to underlie this switch in axonal responses is the silencing of Netrin1/Deleted in Colorectal Carcinoma (DCC)-mediated attraction by the repulsive SLIT/ROBO1 signaling. Using in vivo approaches including CRISPR-Cas9-engineered mouse models of distinct Dcc splice isoforms, we show here that commissural axons maintain responsiveness to both Netrin and SLIT during midline crossing, although likely at quantitatively different levels. In addition, full-length DCC in collaboration with ROBO3 can antagonize ROBO1 repulsion in vivo. We propose that commissural axons integrate and balance the opposing DCC and Roundabout (ROBO) signaling to ensure proper guidance decisions during midline entry and exit.

Netrin-1 inducing antiapoptotic effect of acute myeloid leukemia cells in a concentration-dependent manner through the Unc-5 netrin receptor B-focal adhesion kinase axis.

Acute myeloid leukemia (AML) is a hematological malignancy that commonly occurs in children. The prognosis of pediatric AML is relatively poor, thus threatening the patient's survival. The aberrant expression of the axon guidance factor, netrin-1, is observed in various types of malignancies, and it participates in the proliferation and apoptosis of tumor cells. Herein, we aimed to explore the role of netrin-1 in AML cells. Netrin-1 is highly expressed in AML patients. Proliferation and anti-apoptosis were observed in AML cells treated with netrin-1. The interaction between netrin-1 and Unc-5 netrin receptor B (UNC5B) was detected through coimmunoprecipitation, and UNC5B ribonucleic acid interference restrained the influence of netrin-1 on the AML cells. The phosphorylation of focal adhesion kinase-protein kinase B (FAK-Akt) was upregulated in AML cells treated with netrin-1. Both FAK and Akt inhibitors abrogated the effects of netrin-1 on the proliferation and apoptosis of AML cells. In conclusion, netrin-1 could promote the growth and reduce the apoptosis of AML cells in a concentration-dependent manner, and that these effects were mediated by activating the FAK-Akt signaling pathway via the UNC5B.

Viral delivery of L1CAM promotes axonal extensions by embryonic cerebral grafts in mouse brain.

Cell replacement therapy is expected as a new and more radical treatment against brain damage. We previously reported that transplanted human cerebral organoids extend their axons along the corticospinal tract in rodent brains. The axons reached the spinal cord but were still sparse. Therefore, this study optimized the host brain environment by the adeno-associated virus (AAV)-mediated expression of axon guidance proteins in mouse brain. Among netrin-1, SEMA3, and L1CAM, only L1CAM significantly promoted the axonal extension of mouse embryonic brain tissue-derived grafts. L1CAM was also expressed by donor neurons, and this promotion was exerted in a haptotactic manner by their homophilic binding. Primary cortical neurons cocultured on L1CAM-expressing HEK-293 cells supported this mechanism. These results suggest that optimizing the host environment by the AAV-mediated expression of axon guidance molecules enhances the effect of cell replacement therapy.

An imbalance of netrin-1 and DCC during nigral degeneration in experimental models and patients with Parkinson's disease.

AIMS: Multiple guidance cues, such as netrin-1 (NTN-1)/deleted in colorectal carcinoma (DCC), control the guidance of axons and help establish functional neural circuits during development. However, the function of these guidance molecules during the neurodegenerative process is unclear. METHODS: To access the alterations of NTN-1 and DCC during the onset and progression of PD, we first established two subacute and one chronic PD model. Then, we investigated the relationship between the NTN-1/DCC pathway and cell death in SH-SY5Y cells. Finally, we conducted correlation studies between plasma NTN-1 and parkinsonian symptoms in patients to understand how this pathway contributes to PD. RESULTS: We found that the imbalance of NTN-1 and DCC was a common feature of nigral DA neuron injury in PD mouse models. We investigated that MPP+ inhibited NTN-1 expression and increased DCC expression in a concentration- and time-dependent manner. We further discovered a significant decrease in plasma NTN-1 levels and a positive correlation with UPDRS scores in PD patients. CONCLUSION: Our findings confirmed the imbalance of NTN-1/DCC signaling during nigral degeneration in experimental PD models and found for the first time a correlation of plasma NTN-1 with PD symptoms in patients.