Neuronal guidance genes in health and diseases

Neurons migrate from their birthplaces to the destinations, and extending axons navigate to their synaptic targets by sensing various extracellular cues in spatiotemporally controlled manners. These evolutionally conserved guidance cues and their receptors regulate multiple aspects of neural development to establish the highly complex nervous system by mediating both short- and long-range cell-cell communications. Neuronal guidance genes (encoding cues, receptors, or downstream signal transducers) are critical not only for development of the nervous system but also for synaptic maintenance, remodeling, and function in the adult brain. One emerging theme is the combinatorial and complementary functions of relatively limited classes of neuronal guidance genes in multiple processes, including neuronal migration, axonal guidance, synaptogenesis, and circuit formation. Importantly, neuronal guidance genes also regulate cell migration and cell-cell communications outside the nervous system. We are just beginning to understand how cells integrate multiple guidance and adhesion signaling inputs to determine overall cellular/subcellular behavior and how aberrant guidance signaling in various cell types contributes to diverse human diseases, ranging from developmental, neuropsychiatric, and neurodegenerative disorders to cancer metastasis. We review classic studies and recent advances in understanding signaling mechanisms of the guidance genes as well as their roles in human diseases. Furthermore, we discuss the remaining challenges and therapeutic potentials of modulating neuronal guidance pathways in neural repair.

Protective effects of Slit2 on corneal epithelium and nerves in diabetic mice and its mechanism / 中华实验眼科杂志

Objective:To explore the promoting effects of slit guidance ligand 2 (Slit2) on the repair of corneal epithelium and nerve damage in diabetic mice and possible molecular mechanism.Methods:Sixty SPF C57BL/6 mice aged 5-6 weeks were divided into normal control group, diabetes model group and Slit2 injection group according to the random number table method, 20 for each group.Diabectic model was prepared by intraperitoneal injection of streptozotocin in the diabetes model group and Slit2 injection group.A mouse corneal epithelial injury repair model was established using electric epithelial scraper, and Slit2 recombinant protein was subconjunctivally injected immediately following modeling in the Slit2 injection group.The equal volume of phosphate buffer saline (PBS) was used in a same way in the diabetes model group.No intervention was performed in the normal control group.Corneal epithelial healing were examined at 24, 48 and 72 hours after corneal epithelial defect by corneal fluorescin staining.Real-time fluorescent quantitative PCR was used to detect the expression of Slit2 and its related receptors in the corneal epithelium of normal and diabetic model mice.Fluorescence staining of corneal wholemount with β-tubulin Ⅲ was used to observe the changes in corneal nerve morphology.Immunofluorescence staining was performed to detect the expression and distribution of Slit2 in mouse corneal epithelium in normal control group and diabetes model group, as well as the expression and distribution of Slit2, epidermal growth factor receptor (EGFR), extracellular-signal-regulated kinase (ERK), threonine protein kinase (AKT), β-catenin and Ki67 in the healing corneal epithelium of mice after corneal epithelium damage in different groups.The mouse corneal epithelial stem/progenitor cell line (TKE2) was divided into normal control group, high-glucose group and Slit2 treatment group.Western blot was performed to detect the expression of p-EGFR/EGFR and p-AKT/AKT in the TKE2 of the three groups.The expression of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 with 0.01, 0.1 and 0.5 μg/ml Slit2 treatment for 10 minutes, and before and 10, 20, 30, 60, 120 minutes after 0.5 μg/ml Slit2 treatment was detected by Western blot.The effects of Slit2 on the axon regeneration of mouse trigeminal ganglion cells (TGs) were observed by immunofluorescence staining.The use and care of animals complied with the ARVO statement.This study protocol was approved by an Ethics Committee of Qingdao Eye Hospital of Shandong First Medical University (No.[2020]57).Results:At 48 and 72 hours after corneal epithelial scraping, the speed of corneal epithelial repair was significantly slowed down in diabetes model group in comparison with the normal control group and Slit2 injection group.The relative expression levels of Slit2 and its receptors Robo1, Robo2 and Robo4 mRNA in the normal corneal epithelium in the diabetes model group were significantly higher than those of the normal control group (all at P<0.05). The fluorescence intensity of Slit2 in normal corneal epithelium in diabetes model group was similar to the normal control group, and the fluorescence intensity of Slit2 in damaged corneal epithelium in diabetic mice was significantly weaker than that in normal control group.Corneal nerve plexus was denser at 7 days after corneal epithelial injury and the nerve fibers were increased with more branches in Slit2 injection group compared with diabetic group.The fluorescence intensity of p-EGFR, p-ERK, β-catenin and Ki67 in damaged corneal epithelium in normal control group and Slit2 injection group was stronger than that of the diabetes model group.The relative expression levels of p-EGFR/EGFR, p-AKT/AKT, and β-catenin in TKE2 in high-glucose group were significantly lower than those in normal control group and Slit2 treatment group (all at P<0.05). The relative expression levels of p-EGFR/EGFR and p-AKT/AKT in high glucose-cultured TKE2 after Slit2 treatment were significantly increased in comparison with before Slit2 treatment (both at P<0.05), and the relative expression levels of p-EGFR/EGFR and p-AKT/AKT in TKE2 were elevated as the increase of Slit2 concentration.The activation effect of 0.5 μg/ml Slit2 on EGFR and AKT pathways was most obvious.The synapse length of TGs cultured by high glucose was (40.52±5.44) μm, which was significantly shortened than (72.14±9.48) μm in normal control group and (73.04±4.66) μm in Slit2 injection group (both at P<0.05). Conclusions:Slit2 can protect the corneal epithelium by activating EGFR signaling pathway and play a protective role to neurons by increasing the density of corneal subepithelial plexus and promoting the growth of TGs axons in diabetic mice.

Research progress of acupuncture and moxibustion on axon guidance cues / 针刺研究

Axon guidance cues includes Slit, Semaphorin, Ephrin and Netrin. They have the function of regulating the regeneration of axons and guiding the regenerated axons to the correct target. They can affect the nervous system, cardiovascular system, and participate in the proliferation and migration of tumor cells. The current research on the influence of acupuncture and moxibustion(mainly focusing on electroacupuncture) on axon guidance cues is limited to animal experiments. Electroacupuncture can treat diseases dominated by the nervous system by regulating the expression of axon guidance cues. This review summarizes the research progress of acupuncture and moxibustion on regulating axonal guidance cues, is hoped to provide references for the mechanism of acupuncture and moxibustion in treating nervous system disease and ideas for acupuncture treatment of diseases related to axon guidance cues.

Axon Guidance Molecules Guiding Neuroinflammation

Axon guidance molecules (AGMs), such as Netrins, Semaphorins, and Ephrins, have long been known to regulate axonal growth in the developing nervous system. Interestingly, the chemotactic properties of AGMs are also important in the postnatal period, such as in the regulation of immune and inflammatory responses. In particular, AGMs play pivotal roles in inflammation of the nervous system, by either stimulating or inhibiting inflammatory responses, depending on specific ligand-receptor combinations. Understanding such regulatory functions of AGMs in neuroinflammation may allow finding new molecular targets to treat neurodegenerative diseases, in which neuroinflammation underlies aetiology and progression.

Functions of semaphorin 4D in tumor progression / 中国肿瘤临床

Semaphorin 4D (SEMA4D), also known as CD100, is a protein that belongs to class IV semaphorin. Its physiologic role in the nervous system has been extensively explored. However, the roles of SEMA4D have extended beyond the traditionally studied territories. Collective data proved that SEMA4D has an important role in the regulation of immune system, angiogenesis, and tumorigenesis, among others. Specifically, SEMA4D enhanced the angiogenesis in malignant diseases. This review summarized the latest progression in the research on SEMA4D, including the structure, receptors, mechanism, and biological functions in tumor angiogenesis and progression.

Research expression of Slit3 and Robo4 in corneal neovascularization of rats / 国际眼科杂志(Guoji Yanke Zazhi)

AlM: To explore the roles of neuronal axon-guidance molecules Slit3 and Robo4 receptor in corneal neovascularization ( CNV ) by study their expression in neovascularized cornea of rats. METHODS: CNV models were established by implantation pellets containing basic fibroblast growth factor ( bFGF ) into corneal stroma. CNV models were measured by biomicroscopy photography. lmmunohistochemical staining and imaging analysis system were used to detect the expression of Slit3 and Robo4 in the models after 1, 4, 7, 10 and 14d. RESULTS:The area of CNV and the expression of Slit3, Robo4 were increased in CNV models compared to that in normal cornea and reached highest level on 7d. And the expression level of Slit3 and Robo4 were significantly correlated with the size of CNV on every time point except 1d (r=0. 84-0. 91, all P CONCLUSlON: The expression of Slit3 and Robo4 may be related to the CNV development. They are potential therapeutical target for CNV.

Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury / 中华急诊医学杂志

Objective To observe the expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury (ALI) and explore the function of Slit2 and Robo4 in ALI.Methods Forty-eight Sprague-Dawley rats were randomly (random number) divided into control group (n =24) and ALl group (n =24).ALI model was reproduced by cecum ligation and puncture (CLP).The control group only experienced a simulated operation without CLP.Both groups were further divided into 3 subgroups with 8 rats in each subgroup:12 h,24 h,and 48 h subgroups.artery blood gas analysis,lung tissue wet/dry weight (W/D) ratio,lung histopathologic changes,pulmonary microvascular permeability were observed.The serum tumor nocrosis factor-α (TNF-α) was measured with enzyme linked immunosorbent assay (ELISA).The expression of Slit2 and Robo4 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR).The expression of Slit2 and Robo4 protein in lung tissues was assessed by immunohistochemistry.Date were analyzed by one-way ANOVA with SPSS version 13.0 software.Statistical significance was established at a P value of less than 0.05.Results Compared with the control group,in ALI rats at different time points,partial pressure of oxygen in arterial blood (PaO2) decreased significantly,lung W/D weight ratio and pulmonary microvascular permeability,the serum TNF-α increased significantly (all P ＜ 0.05),histopathology of lung revealed signs of injury.The expression of Slit2 mRNA in lung tissues was decreased markedly after CLP compared with control group [(0.56±0.13) vs.(0.87±0.05),F=41.39,P＜0.05,(0.42±0.10) vs.(0.85±0.07),F=93.54,P＜0.05,(0.26±0.08) vs.(0.89 ±0.09),F=227.05,P＜0.05].but there were no significant difference in expression of Robo4 mRNA in lung tissue between ALI group and control group [(0.86±0.07) vs.(0.83±0.05),F=0.695,P＞0.05,(0.82±0.05) vs.(0.89±0.08),F=2.061,P ＞ 0.05,(0.86 ± 0.08) vs.(0.86 ± 0.05),F =0.035,P ＞ 0.05].Immunohistochemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells,while lung epithelial cell nuclei and endochylema.Robo4 protein was only expressed on the extracellular surface of vascular endothelial cells.Compared with the control group,expression of Slit2 protein in lung tissue in ALI group decreased markedly [(0.37 ± 0.05) vs.(0.45 ± 0.07),F =6.82,P ＜ 0.05,(0.32±0.06) vs.(0.47±0.09),F=23.54,P＜0.05,(0.28±0.07) vs.(0.46±0.06),F=28.01,P ＜ 0.05].As good as RT-PCR,there were no significant difference in expression of Robo4 protein in lung tissue between two groups [(0.53±0.04) vs.(0.52±0.05),F=0.155,P＞0.05,(0.53± 0.09) vs.(0.50±0.05),F=0.498,P＞0.05,(0.55±0.06) vs.(0.56±0.07),F=0.073,P ＞ 0.05].Conclusions Lung tissues of control group rats express Slit2 and Robo4.The decreased Slit2 mRNA and protein expressions in the lung tissue of rat with ALI caused by CLP may be associated with the occurrence of ALI.

Effect of Ephrin-A1/EphA2 on Invasion of Trophoblastic Cells / 华中科技大学学报（医学）（英德文版）

The effect of axon guidance factors ephrin-A 1/EphA2 on the invasion of trophoblastic cells and the possible mechanism were investigated in this study.The expression of EphA2 in vascular endothelial cells was detected by immunohistochemistry.The proliferation and invasion of TEV-1 cells (an extravillous trophoblastic cell line) in first trimester were determined by cell counting kit-8 (CCK-8)and Transwell invasion assay.Real-time PCR was used to detect the expression of ephrin-A1 in TEV-1cells treated with EphA2 at different concentrations (10,50,100,500,1000 and 5000 μg/L).The results showed:(1) EphA2 was expressed in the vascular endothelial cells; (2) EphA2 could promote the proliferation of TEV-1 cells.The proliferative capacity reached a peak in TEV-1 cells treated with 100 μg/L EphA2 (P＜0.05); (3) EphA2 could increase the invasion of TEV-1 cells.The invasive ability was the greatest in TEV-1 cells treated with 500 μg/L EphA2 (P＜0.05); (4) in the presence of EphA2 (0-500μg/L),the expression of ephrin-A1 was increased concentration-dependently (P＜0.05),but when the concentration of EphA2 was over 500 μg/L,the expression of ephrin-A 1 ceased to increase (P＞0.05).It was concluded that EphA2 can promote the invasion and proliferation of the human extravillous trophoblastic cells probably by regulating the ephrin-A1 ligand.

Fyn Tyrosine Kinase-mediated Tyrosine Phosphorylation of Roundabout (Robo), the Slit Receptor / 대한해부학회지

In this study, the molecular mechanism of tyrosine phosphorylation of Roundabout (Robo), the transmembrane receptor for slits, was investigated. The tyrosine phosphorylation of intracellular portion of Robo was increased by the treatment of tyrosine phosphatase inhibitors in human embryonic kidney cells transfected with Robo. The Robo tyrosine phosphorylation was inhibited by the treatment of Src family kinase inhibitor, PP2. The co-transfection of constitutively active form of Fyn, not the dominant negative form of Fyn, and Robo dramatically enhanced the tyrosine phosphorylation of Robo. Furthermore, the SH2 domain of Fyn, which binds to phosphorylated tyrosine residues, interact with Robo, and the interaction was increased by the inhibition of tyrosine phosphatases. These findings indicate that the tyrosine phosphorylation of Robo is regulated by Fyn.

Multiple Regulation of Roundabout (Robo) Phosphorylation in a Heterologous Cell System

Roundabout (Robo) is the transmembrane receptor for slit, the neuronal guidance molecule. In this study, the tyrosine phosphorylation of Robo was observed in Robo-transfected human embryonic kidney cells and developing rat brains, and found to be increased by the treatment with protein kinase A activator, forskolin. In contrast, protein kinase C activation by phorbol-12-myristate-13-acetate decreased the phosphorylation of Robo. Intracellular calcium was required for the tyrosine phosphorylation. Furthermore, the transfection of an Eph receptor tyrosine kinase dramatically enhanced the tyrosine phosphorylation. These findings indicate that the tyrosine phosphorylation of Robo is regulated by multiple mechanisms, and that Eph receptor kinases may play a role in the regulation of tyrosine phosphorylation of Robo in the rat brain.

Hierarchical clustering the genes related to mouse optic nerve development and injury / 第三军医大学学报

Objective To find out the correlation among the genes which were screened by high density genechips related to mice optic nerve development and injury in our previous work,then to estimate the functions of some unknown genes according to some genes known related to CNS neurite extention and guidance.Methods Hierarchical clustering method was applied for the genes mentioned above and the subgroups respectively containing Ptn,Efnb3 were further analyzed.Results A total of 1 033 suitable candidate genes were clustered into 6 groups.The gene expression pattern of group B was identical to the development pattern of lateral geniculate body(LGN),which supposed that the genes of group B might be the key genes to the optic never development.Five functionally unknown genes Mm.28443,Mm.9671,Mm.25504,Mm.160640,Mm.182895 were clustered into a subgroup together with Ptn.The Pearson's coefficient between each of them and Ptn varied from 0.979 to 0.996.These genes were supposed to be candidate genes related to neurite growth and guidance.Lamr1 and its ligand were assumed to be the neurite guiding molecules for optic never,both beacuse of its high Coefficient to Efnb3 gene and related documents.Conclusion New neurite guiding molecules might be potential target genes for CNS regeneration by genetic engineering.

Changes of expressions of axon guidance molecule Sema3F and its receptor Np2 in hippocampus of temporal lobe epilepsy rat / 临床神经病学杂志

Objective To investigate the changes of expressions of axon guidance molecule Sema3F and its receptor Np2 in hippocampus of temporal lobe epilepsy(TLE)rat.Methods Rats were injected with Lithium-chloride,Pilocarpine intraperitoneally to establish TLE model.The Sema3F mRNA,Np2 mRNA and protein in areas CA1,CA3 and dentate gyrus(DG)of hippocampus at different time were detected by immunohistochemistry and in situ hybridization for TLE models.Further,they were compared with normal control group.Results Compared with normal control group,the expressions of Sema3F mRNA,Np2 mRNA and protein in areas CA1,CA3 significantly decreased(P

Recent advances in Eph receptors and Ephrin ligands / 中国癌症杂志

Protein tyrosine kinase genes are the largest family of oncogenes. This is not surprising since protein tyrosine kinases are important components of signal transduction pathways that control cell shapes, proliferation, differentiation, and migration. This review will address the recent progress in understanding the function of Eph receptor and ephrin ligands in normal development and how disregulation of these functions could promote tumorigenesis.