Axon Guidance Cues Research Articles

A novel method for inducing nerve growth via modulation of host resting potential: gap junction-mediated and serotonergic signaling mechanisms.

SummaryA major goal of regenerative medicine is to restore the function of damaged or missing organs through the implantation of bioengineered or donor-derived components. It is necessary to understand the signals and cues necessary for implanted structures to innervate the host, as organs devoid of neural connections provide little benefit to the patient. While developmental studies have identified neuronal pathfinding molecules required for proper patterning during embryogenesis, strategies to initiate innervation in structures transplanted at later times or alternate locations remain limited. Recent work has identified membrane resting potential of nerves as a key regulator of growth cone extension or arrest. Here, we identify a novel role of bioelectricity in the generation of axon guidance cues, showing that neurons read the electric topography of surrounding cells, and demonstrate these cues can be leveraged to initiate sensory organ transplant innervation. Grafts of fluorescently labeled embryological eye primordia were used to produce ectopic eyes in Xenopus laevis tadpoles. Depolarization of host tissues through anion channel activation or other means led to a striking hyperinnervation of the body by these ectopic eyes. A screen of possible transduction mechanisms identified serotonergic signaling to be essential for hyperinnervation to occur, and our molecular data suggest a possible model of bioelectrical control of the distribution of neurotransmitters that guides nerve growth. Together, these results identify the molecular components of bioelectrical signaling among cells that regulates axon guidance, and suggest novel biomedical and bioengineering strategies for triggering neuronal outgrowth using ion channel drugs already approved for human use.Electronic supplementary materialThe online version of this article (doi:10.1007/s13311-014-0317-7) contains supplementary material, which is available to authorized users.

The Role of Netrin-1 in Diabetic Retinopathy: A Promising Therapeutic Strategy

Netrin-1 which belongs to axon guidance cue is necessary for neural and vascular development. It involves in regulating axon guidance for attraction or repulsion and it has a dual function in endothelial tip cell migration during angiogenesis. Many evidences have proved that netrin-1 plays an important role in angiogenesis, cancer progression and inflammation disease. Here we provide a comprehensive review on the role of netrin-1 in retinal and angiogenesis developments .What’s more, some associated signaling pathways in diabetic retinopathy are also discussed. Overall, the current available data suggests that the netrin-1 could be a promising target for the development of antiangiogenesis drugs.

Engrailed homeoproteins in visual system development.

Engrailed is a homeoprotein transcription factor. This family of transcription factors is characterized by their DNA-binding homeodomain and some members, including Engrailed, can transfer between cells and regulate protein translation in addition to gene transcription. Engrailed is intimately involved in the development of the vertebrate visual system. Early expression of Engrailed in dorsal mesencephalon contributes to the development and organization of a visual structure, the optic tectum/superior colliculus. This structure is an important target for retinal ganglion cell axons that carry visual information from the retina. Engrailed regulates the expression of Ephrin axon guidance cues in the tectum/superior colliculus. More recently it has been reported that Engrailed itself acts as an axon guidance cue in synergy with the Ephrin system and is proposed to enhance retinal topographic precision.

The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP.

UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. We previously discovered that attractive UNC-40/DCC receptor signaling stimulates growth cone filopodial protrusion and that repulsive UNC-40–UNC-5 heterodimers inhibit filopodial protrusion in C. elegans. Here, we identify cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. We show that the Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition.

Involvement of Sema4A in multiple sclerosis and neuromyelitis optica

Semaphorins were initially identified as axon guidance cues during neural development, however, several semaphorins are crucially involved in various phases of immune responses. Sema4A is one of the membrane-type class IV semaphorins, which is expressed in dendritic cells (DCs) and plays critical roles in T cell activation during the course of experimental autoimmune encephalomyelitis; however, its pathological involvement in human inflammatory demyelinating diseases has not been determined. Here, we report that serum Sema4A is increased in approximately one-third of patients with Multiple Sclerosis (MS) and Neuromyelitis Optica (NMO).

Prediction of horseshoe configuration in neural receptors: Dscam isoforms

Dscam (Down Syndrome Cell Adhesion Molecule), a member of immunoglobulin superfamily (IgSF), is a receptor for netrin-1 [1,2], a classical axon guidance cue. The interaction between netrin-1 and Dscam plays an important role in the development of the nervous system. Here, we present the crystal structure of the N-terminal four Ig-like domains of Dscam. The molecule folds into a horseshoe-like configuration. A comparison of this Dscam horseshoe with the previously described horseshoe structures of other cell-surface receptors has revealed fascinating conserved structural features and important sequence markers for these IgSF proteins. This discovery can help us predict the horseshoe configuration N-terminal arrangements in a number of other structurally unknown neural receptors. The N-terminal horseshoe has been shown to be engaged in homophilic and heterophilic interactions between two cell-surface receptors.

Complementary expression of EphA7 and SCO-spondin during posterior commissure development.

Bilaterally symmetric organisms need to exchange information between the two sides of their bodies in order to integrate sensory inputs and coordinate motor control. This exchange occurs through commissures formed by neurons that project axons across the midline to the contralateral side of the central nervous system. The posterior commissure is the first transversal axonal tract of the embryonic vertebrate brain. It is located in the dorsal portion of the prosomere 1, at the caudal diencephalon. The axons of the posterior commissure principally come from neurons of ventrolateral and dorsolateral pretectal nuclei (parvocellular and magnocellular nucleus of the posterior commissure, respectively) that extend their axons toward the dorsal region. The trajectory of these axons can be divided into the following three stages: (1) dorsal axon extension towards the lateral roof plate; (2) fasciculation in the lateral roof plate; and (3) midline decision of turning to the ipsilateral side or continuing to the opposite side. The mechanisms and molecules that guide the axons during these steps are unknown. In the present work, immunohistochemical and in situ hybridization analyses were performed, with results suggesting the participation of EphA7 in guiding axons from the ventral to the dorsal region of the prosomere 1 through the generation of an axonal corridor limited by repulsive EphA7 walls. At the lateral roof plate, the axons became fasciculated in presence of SCO-spondin until reaching the midline. Finally, EphA7 expression was observed in the diencephalic midline roof plate, specifically in the region where some axons turn to the ipsilateral side, suggesting its participation in this decision. In summary, the present work proposes a mechanism of posterior commissure formation orchestrated by the complementary expression of the axon guidance cues SCO-spondin and EphA7.

Netrin-1 controls sympathetic arterial innervation

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

ADAM metalloproteases promote a developmental switch in responsiveness to the axonal repellant Sema3A.

During embryonic development, axons can gain and lose sensitivity to guidance cues, and this flexibility is essential for the correct wiring of the nervous system. Yet, the underlying molecular mechanisms are largely unknown. Here we show that receptor cleavage by ADAM (A Disintegrin And Metalloprotease) metalloproteases promotes murine sensory axons loss of responsiveness to the chemorepellant Sema3A. Genetic ablation of ADAM10 and ADAM17 disrupts the developmental downregulation of Neuropilin-1 (Nrp1), the receptor for Sema3A, in sensory axons. Moreover, this is correlated with gain of repulsive response to Sema3A. Overexpression of Nrp1 in neurons reverses axonal desensitization to Sema3A, but this is hampered in a mutant Nrp1 with high susceptibility to cleavage. Lastly, we detect guidance errors of proprioceptive axons in ADAM knockouts that are consistent with enhanced response to Sema3A. Our results provide the first evidence for involvement of ADAMs in regulating developmental switch in responsiveness to axonal guidance cues.

FRI0512 Involvement of Plexind1/Semaphorin 3E Pathway in the Dysregulation of Vascular Tone Control in Systemic Sclerosis Patients

BackgroundThe main hallmark of systemic sclerosis (SSc) is vasculopathy characterised by dysregulation of angiogenesis and vascular tone leading to loss of capillaries. The vascular and nervous system have several anatomic...

Inhibitory effects of draxin on axonal outgrowth and migration of precerebellar neurons

The rhombic lip, a dorsal stripe of the neuroepithelium lining the edge of the fourth ventricle, is the site of origin of precerebellar neurons (PCN), which migrate tangentially towards the floor plate. After reaching the floor plate, they project their axons to the cerebellum. Although previous studies have shown that the guidance molecules Netrin/DCC and Slit/Robo have critical roles in PCN migration, the molecular mechanisms underlying this process remain poorly understood. Here, we report that draxin, a repulsive axon guidance protein, is involved in PCN development. We found that draxin is expressed in the rhombic lip and migratory stream of some PCN in the developing hindbrain of mice. In addition, draxin inhibited neurite outgrowth and nuclei migration from rhombic lip explants. These results suggest that draxin functions as a repulsive guidance cue for PCN migration. However, we observed no significant differences in PCN distribution between draxin−/− and wild type embryos. Thus, draxin and other axon guidance cues may have redundant roles in PCN migration.

Human neural stem cells survive long term in the midbrain of dopamine-depleted monkeys after GDNF overexpression and project neurites toward an appropriate target.

Transplanted multipotent human fetal neural stem cells (hfNSCs) significantly improved the function of parkinsonian monkeys in a prior study primarily by neuroprotection, with only 3%-5% of cells expressing a dopamine (DA) phenotype. In this paper, we sought to determine whether further manipulation of the neural microenvironment by overexpression of a developmentally critical molecule, glial cell-derived neurotrophic factor (GDNF), in the host striatum could enhance DA differentiation of hfNSCs injected into the substantia nigra and elicit growth of their axons to the GDNF-expressing target. hfNSCs were transplanted into the midbrain of 10 green monkeys exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydro-pyridine. GDNF was delivered concomitantly to the striatum via an adeno-associated virus serotype 5 vector, and the fate of grafted cells was assessed after 11 months. Donor cells remained predominantly within the midbrain at the injection site and sprouted numerous neurofilament-immunoreactive fibers that appeared to course rostrally toward the striatum in parallel with tyrosine hydroxylase-immunoreactive fibers from the host substantia nigra but did not mature into DA neurons. This work suggests that hfNSCs can generate neurons that project long fibers in the adult primate brain. However, in the absence of region-specific signals and despite GDNF overexpression, hfNSCs did not differentiate into mature DA neurons in large numbers. It is encouraging, however, that the adult primate brain appeared to retain axonal guidance cues. We believe that transplantation of stem cells, specifically instructed ex vivo to yield DA neurons, could lead to reconstruction of some portion of the nigrostriatal pathway and prove beneficial for the parkinsonian condition.

Different roles of axon guidance cues and patterned spontaneous activity in establishing receptive fields in the mouse superior colliculus.

Visual neurons in the superior colliculus (SC) respond to both bright (On) and dark (Off) stimuli in their receptive fields. This receptive field property is due to proper convergence of On- and Off-centered retinal ganglion cells to their target cells in the SC. In this study, we have compared the receptive field structure of individual SC neurons in two lines of mutant mice that are deficient in retinotopic mapping: the ephrin-A knockouts that lack important retinocollicular axonal guidance cues and the nAChR-β2 knockouts that have altered activity-dependent refinement of retinocollicular projections. We find that even though the receptive fields are much larger in the ephrin-A knockouts, their On–Off overlap remains unchanged. These neurons also display normal level of selectivity for stimulus direction and orientation. In contrast, the On–Off overlap is disrupted in the β2 knockouts. Together with the previous finding of disrupted direction and orientation selectivity in the β2 knockout mice, our results indicate that molecular guidance cues and activity-dependent processes play different roles in the development of receptive field properties in the SC.

ENU-3 is a new player in UNC-6/Netrin directed axon outgrowth and guidance in C. elegans

UNC-6/Netrin is an axon guidance cue that works through UNC-40/DCC/Frazzled to attract cells and neurons and through UNC-5 and UNC-40 to repulse cells and neurons during nervous system development. The gene encoding the novel protein ENU-3 was identified in a mutant that enhanced the axon outgrowth defects of the DA and DB classes of motor neurons in a strain lacking functional UNC-5. Mutant ENU-3 also enhanced the axon outgrowth and guidance defects of the ventrally directed processes of the AVM and PVM touch receptor neurons in a strain lacking functional UNC-40. ENU-3 also plays a role in adhesion of axons to the substratum as does UNC-40. The protein is a putative single pass transmembrane protein expressed throughout the nervous system whose possible biochemical roles are currently unknown.

Expression of axon guidance cues Slit2 and Robo4 in lung tissue of rat with acute lung injury

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. MethodsForty-eight Sprague-Dawley rats were randomly （random number） divided into control group （n = 24） and ALI 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 ]. Immunohistoehemistry study showed Slit2 protein was mainly expressed on the extracellular surface of vascular endothelial cells, while lung epithelial cell nuclei and endoehylema. 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 ALl 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 ALl caused by CLP may be associated with the occurrence of ALl. Key words: Slit2; Robo4; Acute lung injury; Sepsis; Endothelial permeability; Cecum ligation and puncture; TNF-α; Axon guidance cues

FLRT3 Is a Robo1-Interacting Protein that Determines Netrin-1 Attraction in Developing Axons

Guidance molecules are normally presented to cells in an overlapping fashion; however, little is known about how their signals are integrated to control the formation of neural circuits. In the thalamocortical system, the topographical sorting of distinct axonal subpopulations relies on the emergent cooperation between Slit1 and Netrin-1 guidance cues presented by intermediate cellular targets. However, the mechanism by which both cues interact to drive distinct axonal responses remains unknown. Here, we show that the attractive response to the guidance cue Netrin-1 is controlled by Slit/Robo1 signaling and by FLRT3, a novel coreceptor for Robo1. While thalamic axons lacking FLRT3 are insensitive to Netrin-1, thalamic axons containing FLRT3 can modulate their Netrin-1 responsiveness in a context-dependent manner. In the presence ofSlit1, both Robo1 and FLRT3 receptors are required to induce Netrin-1 attraction by the upregulation of surface DCC through the activation of protein kinase A. Finally, the absence of FLRT3 produces defects in axon guidance invivo. These results highlight a novel mechanism by which interactions between limited numbers of axon guidance cues can multiply the responses in developing axons, as required for proper axonal tract formation in the mammalian brain.

Developmental Gene Expression Profile of Axon Guidance Cues in Purkinje Cells During Cerebellar Circuit Formation

The establishment of precise neural circuits during development involves a variety of contact-mediated and secreted guidance molecules that are expressed in a complementary fashion by different cell types. To build a functional circuit, each cell type must first trigger an intrinsic genetic program that is led by their environment at a key time point. It is therefore essential to identify the different cell-specific and stage-specific transcriptional profiles expressed by neurons. However, very few studies have been done to address this issue thus far. Herein, we have carried out a large-scale quantitative real-time PCR analysis of all classical axon guidance molecules (i.e., Semaphorins, Netrins, Ephrins, and Slits) and their receptors expressed by Purkinje cells (PCs) at specific stages of postnatal cerebellar development in vivo. Most cerebellar connections are setup in a well-characterized sequential manner during postnatal development and lead to the fine regulation of the PC, the sole output of the structure. Our analysis of the relative expression of these guidance cues has uncovered a dynamic expression pattern corresponding to specific stages of cerebellar development, thus providing a starting point for studying the role of these axon guidance molecules in cerebellar wiring.

Down-Regulation of UNC5D in Bladder Cancer: UNC5D as a Possible Mediator of Cisplatin Induced Apoptosis in Bladder Cancer Cells

Identifying potential targets would improve therapeutic planning anddisease management. Therefore, we investigated whether the novel identified dependence receptor UNC5D acts as a tumor suppressor in bladder malignancies. We assessed the UNC5D level in a panel of 15 primary bladder carcinomas and 6 cell lines using real-time reverse transcriptase-polymerase chain reaction and Western blot. MTT assay, TUNEL staining, colony formation assay and Western blot were done in cells untransfected and transfected with UNC5D vector, siUNC5D or siDAPK. UNC5D was dramatically down-regulated in bladder cancer tissue samples and malignant cell lines. Restoration of UNC5D expression in bladder cancer cells lacking endogenous UNC5D expression suppressed cell proliferation and survival. Cisplatin treatment significantly induced UNC5D expression and DAPK dephosphorylation while UNC5D knockdown decreased bladder cancer cell sensitivity to cisplatin. DAPK silencing significantly inhibited the effect of UNC5D on apoptosis induced by cisplatin. Our study suggests that UNC5D may have important roles as a novel suppressor in bladder cancer via the UNC5D/DAPK pathway.

Ephrin-B2/EphA4 forward signaling is required for regulation of radial migration of cortical neurons in the mouse

Postmitotic neurons in the neocortex migrate to appropriate positions and form layered structures of nascent cortex during brain development. The migration of these neurons requires precise control and coordination of a large number of molecules such as axon guidance cues. The Eph-ephrin signaling pathway plays important roles in the development of the nervous system in a wide variety of ways, including cell segregation, axon pathfinding, and neuron migration. However, the role of ephrin-B2/EphA4 signaling in cortical neuron migration remains elusive. Here we demonstrated that ephrin-B2 and its receptor EphA4 were expressed in complementary and overlapping patterns in the developing neocortex. Deletion of the EphA4 gene in the embryonic cerebral cortex resulted in faster migration of cortical neurons, whereas knockdown or overexpression of ephrin-B2 did not alter the normal process of migration. These results suggest that ephrin-B2 forward signaling through EphA4 is required for the precise control of cortical neuron migration.

Analysis of calcium signals in steering neuronal growth cones in vitro.

Calcium imaging allows us to measure the spatial and temporal changes in intracellular calcium concentration in living cells. Localized calcium elevation often functions as the polarizing signal during guided migration including axon guidance. In this chapter, we describe a protocol to quantitatively monitor the spatiotemporal dynamics of calcium signals in neuronal growth cones in the presence of an extracellular concentration gradient of axon guidance cue.