Netrin-1 acts as a survival factor via its receptors UNC5H and DCC

The membrane receptors DCC and UNC5H have been shown to be crucial for axon guidance and neuronal migration by acting as receptors for netrin-1. DCC has also been proposed as a dependence receptor inducing apoptosis in cells that are beyond netrin-1 availability. Here we show that the netrin-1 receptors UNC5H (UNC5H1, UNC5H2, UNC5H3) also act as dependence receptors. UNC5H receptors induce apoptosis, but this effect is blocked in the presence of netrin-1. Moreover, we demonstrate that UNC5H receptors are cleaved in vitro by caspase in their intracellular domains. This cleavage may lead to the exposure of a fragment encompassing a death domain required for cell death induction in vivo. Finally, we present evidence that during development of the nervous system, the presence of netrin-1 is crucial to maintain survival of UNC5H- and DCC-expressing neurons, especially in the ventricular zone of the brainstem. Altogether, these results argue for a role of netrin-1 during the development of the nervous system, not only as a guidance cue but as a survival factor via its receptors DCC and UNC5H.     

The dependence receptors DCC and UNC5H as a link between neuronal guidance and survival

Netrins are secreted proteins that play a crucial role in neuronal migration and in axon guidance during the development of the nervous system. Netrin-1 has been shown to interact with the transmembrane receptors DCC and UNC5H and these receptors appeared of key importance in mediating the chemotropic activity of netrin-1. Before the discovery of DCC as a netrin-1 receptor, the gene encoding DCC was proposed as a putative tumor suppressor gene because one DCC allele was deleted in roughly 70% of colorectal cancers and its expression was often reduced or absent in colorectal cancer tissues. A putative explanation for such dual roles has recently emerged with the observation that DCC belongs to the growing family of dependence receptors. Such receptors share the property of inducing apoptosis in the absence of ligand, hence creating a cellular state of dependence on the ligand. The other netrin-1 receptors UNC5H were also subsequently proposed to be dependence receptors, suggesting that netrin-1 may not only be a chemotropic factor for neurons but also a survival factor. We describe here netrin-1 and its receptors, together with the molecular signaling pathways initiated upon netrin-1 binding or upon netrin-1 withdrawal leading respectively to axonal/neuronal guidance or cell death induction. We then conclude to the possible roles of DCC and UNC5H pro-apoptotic activities in both nervous system development and tumorigenesis.     

Netrin-1 mediates neuronal survival through PIKE-L interaction with the dependence receptor UNC5B

Netrins, a family of secreted molecules, have critical functions in axon guidance and cell migration during neuronal development. In addition to its role as a chemotropic molecule, netrin-1 also acts as a survival factor. Both UNC5 (that is, UNC5A, UNC5B, UNC5C or UNC5D) and DCC are transmembrane receptors for netrin-1 (Refs 8, 9). In the absence of netrin-1, DCC and UNC5 act as dependence receptors and trigger apoptosis. However, how netrin-1 suppresses the apoptotic activity of the receptors remains elusive. Here we show that netrin-1 induces interaction of UNC5B with the brain-specific GTPase PIKE-L. This interaction triggers the activation of PtdIns-3-OH kinase signalling, prevents UNC5B's pro-apoptotic activity and enhances neuronal survival. Moreover, this process relies strongly on Fyn because PIKE-L is tyrosine phosphorylated in response to netrin-1, and the netrin-1-mediated interaction of UNC5B with PIKE-L is inhibited in Fyn-null mice. Thus, PIKE-L acts as a downstream survival effector for netrin-1 through UNC5B in the nervous system.     

A ligand-gated association between cytoplasmic domains of UNC5 and DCC family receptors converts netrin-induced growth cone attraction to repulsion.

Netrins are bifunctional: they attract some axons and repel others. Netrin receptors of the Deleted in Colorectal Cancer (DCC) family are implicated in attraction and those of the UNC5 family in repulsion, but genetic evidence also suggests involvement of the DCC protein UNC-40 in some cases of repulsion. To test whether these proteins form a receptor complex for repulsion, we studied the attractive responses of Xenopus spinal axons to netrin-1, which are mediated by DCC. We show that attraction is converted to repulsion by expression of UNC5 proteins in these cells, that this repulsion requires DCC function, that the UNC5 cytoplasmic domain is sufficient to effect the conversion, and that repulsion can be initiated by netrin-1 binding to either UNC5 or DCC. The isolated cytoplasmic domains of DCC and UNC5 proteins interact directly, but this interaction is repressed in the context of the full-length proteins. We provide evidence that netrin-1 triggers the formation of a receptor complex of DCC and UNC5 proteins and simultaneously derepresses the interaction between their cytoplasmic domains, thereby converting DCC-mediated attraction to UNC5/DCC-mediated repulsion.     

Down Syndrome Cell Adhesion Molecule (DSCAM) Associates with Uncoordinated-5C (UNC5C) in Netrin-1-mediated Growth Cone Collapse

In the developing nervous system, neuronal growth cones explore the extracellular environment for guidance cues, which can guide them along specific trajectories toward their targets. Netrin-1, a bifunctional guidance cue, binds to deleted in colorectal cancer (DCC) and DSCAM mediating axon attraction, and UNC5 mediating axon repulsion. Here, we show that DSCAM interacts with UNC5C and this interaction is stimulated by netrin-1 in primary cortical neurons and postnatal cerebellar granule cells. DSCAM partially co-localized with UNC5C in primary neurons and brain tissues. Netrin-1 induces axon growth cone collapse of mouse cerebellum external granule layer (EGL) cells, and the knockdown of DSCAM or UNC5C by specific shRNAs or blocking their signaling by overexpressing dominant negative mutants suppresses netrin-1-induced growth cone collapse. Similarly, the simultaneous knockdown of DSCAM and UNC5C also blocks netrin-1-induced growth cone collapse in EGL cells. Netrin-1 increases tyrosine phosphorylation of endogenous DSCAM, UNC5C, FAK, Fyn, and PAK1, and promotes complex formation of DSCAM with these signaling molecules in primary postnatal cerebellar neurons. Inhibition of Src family kinases efficiently reduces the interaction of DSCAM with UNC5C, FAK, Fyn, and PAK1 and tyrosine phosphorylation of these proteins as well as growth cone collapse of mouse EGL cells induced by netrin-1. The knockdown of DSCAM inhibits netrin-induced tyrosine phosphorylation of UNC5C and Fyn as well as the interaction of UNC5C with Fyn. The double knockdown of both receptors abolishes the induction of Fyn tyrosine phosphorylation by netrin-1. Our study reveals the first evidence that DSCAM coordinates with UNC5C in netrin-1 repulsion.     

The dependence receptor UNC5H2 mediates apoptosis through DAP-kinase

Netrin-1 receptors UNC5H (UNC5H1-4) were originally proposed to mediate the chemorepulsive activity of netrin-1 during axonal guidance processes. However, UNC5H receptors were more recently described as dependence receptors and, as such, able to trigger apoptosis in the absence of netrin-1. They were also proposed as putative tumor suppressors. Here, we show that UNC5H2 physically interacts with the serine/threonine kinase death-associated protein kinase (DAP-kinase) both in cell culture and in embryonic mouse brains. This interaction occurs in part through the respective death domains of UNC5H2 and DAP-kinase. Moreover, part of UNC5H2 proapoptotic activity occurs through this interaction because UNC5H2-induced cell death is partly impaired in the presence of dominant-negative mutants of DAP-kinase or in DAP-kinase mutant murine embryonic fibroblast cells. In the absence of netrin-1, UNC5H2 reduces DAP-kinase autophosphorylation on Ser308 and increases the catalytic activity of the kinase while netrin-1 blocks UNC5H2-dependent DAP-kinase activation. Thus, the pair netrin-1/UNC5H2 may regulate cell fate by controlling the proapoptotic kinase activity of DAP-kinase.     

Tyrosine phosphorylation of netrin receptors in netrin-1 signaling

Deleted in colorectal cancer (DCC) and neogenin are receptors of netrins, a family of guidance cues that promote axon outgrowth and guide growth cones in developing nervous system. The intracellular mechanisms of netrins, however, remain elusive. In this paper, we show that both DCC and neogenin become tyrosine phosphorylated in cortical neurons in response to netrin-1. Using a site-specific antiphosphor DCC antibody, we show that Y1420 phosphorylation is increased in netrin-1-stimulated neurons and that tyrosine-phosphorylated DCC is located in growth cones. In addition, we show that tyrosine-phosphorylated DCC selectively interacts with the Src family kinases Fyn and Lck, but not Src, c-Abl, Grb2, SHIP1, Shc, or tensin, suggesting a role of Fyn or Lck in netrin-1-DCC signaling. Of interest to note is that tyrosine-phosphorylated neogenin and uncoordinated 5 H2 (Unc5H2) not only bind to the Src homology 2 (SH2) domains of Fyn and SHP2, but also interact with the SH2 domain of SHIP1, suggesting a differential signaling between DCC and neogenin/Unc5H2. Furthermore, we demonstrate that inhibition of Src family kinase activity attenuated netrin-1-induced neurite outgrowth. Together, these results suggest a role of Src family kinases and tyrosine phosphorylation of netrin-1 receptors in regulating netrin-1 function.     

The activation of 5-HT receptors in prefrontal cortex enhances dopaminergic activity

Atypical antipsychotics show preferential 5-HT 2A versus dopamine (DA) D2 receptor affinity. At clinical doses, they fully occupy cortical 5-HT2 receptors, which suggests a strong relationship with their therapeutic action. Half of the pyramidal neurones in the medial prefrontal cortex (mPFC) express 5-HT 2A receptors. Also, neurones excited through 5-HT 2A receptors project to the ventral tegmental area (VTA). We therefore hypothesized that prefrontal 5-HT 2A receptors can modulate DA transmission through excitatory mPFC-VTA inputs. In this study we used single unit recordings to examine the responses of DA neurones to local (in the mPFC) and systemic administration of the 5-HT 2A/2C agonist 1-[2,5-dimethoxy-4-iodophenyl-2-aminopropane] (DOI). Likewise, using microdialysis, we examined DA release in the mPFC and VTA (single/dual probe) in response to prefrontal and systemic drug administration. The local (in the mPFC) and systemic administration of DOI increased the firing rate and burst firing of DA neurones and DA release in the VTA and mPFC. The increase in VTA DA release was mimicked by the electrical stimulation of the mPFC. The effects of DOI were reversed by M100907 and ritanserin. These results indicate that the activity of VTA DA neurones is under the excitatory control of 5-HT 2A receptors in the mPFC. These observations may help in the understanding of the therapeutic action of atypical antipsychotics.     

Autocrine netrin function inhibits glioma cell motility and promotes focal adhesion formation

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.     

Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth

Netrin-1 attracts or repels growing axons during development. The UNC5 receptors mediate the repulsive response, either alone or in complex with DCC receptors. The signaling mechanisms activated by UNC5 are poorly understood. Here, we examined the role of Rho GTPases in UNC5a signaling. We found that UNC5a induced neurite outgrowth in N1E-115 neuroblastoma cells in a netrin-1- and Rac1-dependent manner. UNC5a lacking its cytoplasmic tail also mediated this effect. In fibroblasts, UNC5a was able to activate RhoA and to a lower extent Rac1 and Cdc42 in response to netrin-1. Using Fluorescence Resonance Energy Transfer (FRET) intermolecular probes, we visualized the spatial and temporal activation of Rac1, Cdc42 and RhoA in live N1E-115 cells expressing UNC5a during neurite outgrowth. We found that Rac1 but not Cdc42 was transiently activated at the leading edge of the cell during neurite initiation. However, at later times when well-developed neurites were formed, active RhoA was found in the cell body and at the base of the neuronal leading process in UNC5a-expressing cells. Together, these findings demonstrate that the netrin-1 receptor UNC5a is able to induce neurite outgrowth and to differentially activate RhoA and Rac1 during neurite extension in a spatial and temporal manner.     

UNC5H1 induces apoptosis via its juxtamembrane region through an interaction with NRAGE

The UNC5Hs are axon guidance receptors that mediate netrin-1-dependent chemorepulsion, and dependence receptors that mediate netrin-1-independent apoptosis. Here, we report an interaction between UNC5H1 and NRAGE. Our experiments show that this interaction is responsible for apoptosis induced by UNC5H1, and this level of apoptosis is greater than the amount induced by either UNC5H2 or UNC5H3. We mapped the NRAGE binding domain of UNC5H1 to its ZU-5 domain and show that this region, in addition to an adjacent PEST sequence, is required for UNC5H1-mediated apoptosis. Chimeric UNC5H2 and UNC5H3 receptors, containing the NRAGE binding domain and PEST sequence of UNC5H1, bind NRAGE and cause increased levels of apoptosis. UNC5H1 expression does not induce apoptosis in differentiated PC12 cells, which down-regulate NRAGE, but induces apoptosis in native PC12 cells that endogenously express high levels of NRAGE and in differentiated PC12 cells when NRAGE is overexpressed. Together, these results demonstrate a mechanism for UNC5H1-mediated apoptosis that requires an interaction with the MAGE protein NRAGE.     

The dependence receptor UNC5H2/B triggers apoptosis via PP2A-mediated dephosphorylation of DAP kinase

The UNC5H dependence receptors promote apoptosis in the absence of their ligand, netrin-1, and this is important for neuronal and vascular development and for limitation of cancer progression. UNC5H2 (also called UNC5B) triggers cell death through the activation of the serine-threonine protein kinase DAPk. While performing a siRNA screen to identify genes implicated in UNC5H-induced apoptosis, we identified the structural subunit PR65β of the holoenzyme protein phosphatase 2A (PP2A). We show that UNC5H2/B recruits a protein complex that includes PR65β and DAPk and retains PP2A activity. PP2A activity is required for UNC5H2/B-induced apoptosis, since it activates DAPk by triggering its dephosphorylation. Moreover, netrin-1 binding to UNC5H2/B prevents this effect through interaction of the PP2A inhibitor CIP2A to UNC5H2/B. Thus we show here that, in the absence of netrin-1, recruitment of PP2A to UNC5H2/B allows the activation of DAPk via a PP2A-mediated dephosphorylation and that this mechanism is involved in angiogenesis regulation.     

Effects of Chronic Ethanol Consumption and Aging on Dopamine, Serotonin, and Metabolites

Abstract: This study examined the hypothesis that chronic ethanol consumption results in significant abnormalities in both the dopaminergic and the serotonergic system of aged rats. Levels of dopamine (DA), serotonin [5-hydroxytryptamine (5-HT)], 3,4-dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindole-3-acetic acid (5-HIAA) were determined in brain areas of both the nigrostriatal and mesocorticolimbic DA systems in 5-, 14-, and 24-month-old male Fischer 344 rats. Aging was associated with a reduced concentration of DA in the striatum (ST), ventral tegmental area (VTA), and ventral pallidum (VP) and an increased concentration of 5-HIAA in the ST, globus pallidus, nucleus accumbens, frontal cortex, and VP. In addition, there was an increase in the 5-HIAA/5-HT ratio in all brain areas analyzed. Six weeks of ethanol consumption was accompanied by significant changes in mesocorticolimbic brain areas. In the VTA of 5-month-old ethanol-fed rats DA content was decreased to the levels found in aged rats, e.g., 24 months of age. Ethanol also significantly lowered 5-HT and 5-HIAA contents in the VTA and reduced DOPAC and 5-HIAA levels in the VP. In addition, ethanol blunted the normal age-related increase in 5-HIAA/5-HT ratio in the VTA, VP, and substantia nigra. It is interesting that although the age-related changes were found in both nigrostriatal and mesocorticolimbic brain areas, the ethanol-associated effects were found only in brain areas of the mesocorticolimbic system. The changes in DA and 5-HT function that accompany aging and ethanol consumption may contribute to the problems in motor function and ethanol abuse found in the aged.     

Netrin-1: An emerging player in inflammatory diseases

Netrin-1 is a member of the laminin-like protein family and was initially identified as a potent chemotactic molecule involved in axonal guidance and cell migration during embryonic development. Many studies have focused on the non-neural effects of netrin-1, and the results revealed that netrin-1 may be extensively involved in the regulation of angiogenesis, inflammation, tissue remodeling, and cancer. The pathogenic or protective effect of netrin-1 suggests that it may be a potential therapeutic target in multiple diseases. Netrin-1 plays different roles by interacting with its receptors, such as deleted in colorectal cancer (DCC)/neogenin and the uncoordinated-5 homolog family members (UNC5). Interestingly, contradictory actions in certain physiological pathways serve to highlight its manifold and often opposite effects on numerous physiological and pathophysiological processes. Netrin-1 regulates inflammation and leukocyte infiltration, suggesting roles for netrin-1 in the immune response. In this study, we review recent advances in the understanding of netrin-1 and its receptors in many inflammatory diseases and look forward to the bioavailability of netrin-1 for the future.     

UNC5B receptor deletion exacerbates DSS‐induced colitis in mice by increasing epithelial cell apoptosis

The netrin-1 administration or overexpression is known to protect colon from acute colitis. However, the receptor that mediates netrin-1 protective activities in the colon during colitis remains unknown. We tested the hypothesis that UNC5B receptor is a critical mediator of protective function of netrin-1 in dextran sodium sulfate (DSS)-induced colitis using mice with partial deletion of UNC5B receptor. DSS colitis was performed in mice with partial genetic UNC5B deficiency (UNC5B^+/− mice) or wild-type mice to examine the role of endogenous UNC5B. These studies were supported by in vitro models of DSS-induced apoptosis in human colon epithelial cells. WT mice developed colitis in response to DSS feeding as indicated by reduction in bw, reduction in colon length and increase in colon weight. These changes were exacerbated in heterozygous UNC5B knockout mice treated with DSS. Periodic Acid-Schiff stained section shows damages in colon epithelium and mononuclear cell infiltration in WT mice, which was further increased in UNC5B heterozygous knockout mice. This was associated with large increase in inflammatory mediators such as cytokine and chemokine expression and extensive apoptosis of epithelial cells in heterozygous knockout mice as compared to WT mice. Overexpression of UNC5B human colon epithelial cells suppressed DSS-induced apoptosis and caspase-3 activity. Moreover, DSS induced large amount of netrin-1 and shRNA mediated knockdown of netrin-1 induction exacerbated DSS-induced epithelial cell apoptosis. Our results suggest that UNC5B is a critical mediator of cell survival in response to stress in colon.     

Lipid raft localization and palmitoylation: identification of two requirements for cell death induction by the tumor suppressors UNC5H

UNC5H receptors (UNC5H1, UNC5H2, UNC5H3) are putative tumor suppressors whose expression is lost in numerous cancers. These receptors have been shown to belong to the so-called family of dependence receptors. Such receptors induce apoptosis when their ligand netrin-1 is absent, thus conferring a state of cellular dependence towards ligand presence. Along this line, these receptors may limit tumor progression because they induce the death of tumor cells that grow in settings of ligand unavailability. We show here that UNC5H receptors are localized to cholesterol-and sphingolipid-enriched membrane domains called lipid rafts. We then demonstrate that the lipid raft localization of UNC5H2 is required for the pro-apoptotic activity of unbound UNC5H2. We also propose that this lipid raft localization is probably mediated via the recruitment of adaptor protein(s) within the death domain of UNC5H2 but is not dependent on the post-translational modification by palmitoylation of UNC5H2 even though this palmitoylation is required for UNC5H2 pro-apoptotic activity. Moreover we show that the interaction of UNC5H2 with the downstream pro-apoptotic serine threonine kinase DAPk is dependent on both UNC5H2 lipid raft localization and palmitoylation. Thus, we propose that the UNC5H dependence receptors require lipid raft localization and palmitoylation to trigger apoptosis.     

Expression of Netrin-1 and its two receptors DCC and UNC5H2 in the developing mouse lung

The ligand Netrin-1 and its receptors DCC and UNC5H2 are critical for the regulation of neuronal migration in nervous system development. Here we demonstrate expression of these molecules in lung development. The mRNA expression profiles of Netrin-1, DCC and UNC5H2 are developmentally regulated during embryonic mouse lung formation. Netrin-1 shows a bimodal expression pattern with elevated mRNA levels early followed by a second peak in late gestation. Peak expression of DCC occurs early in development whereas expression of UNC5H2 peaks late in development. We also demonstrate localization of Netrin-1, DCC and UNC5H2 during the stages of lung development. We present evidence that these proteins are modulated spatially in the mesenchyme and epithelium during lung organogenesis.