The guidance receptor neogenin promotes pulmonary inflammation during lung injury

Lung injury is marked by a persistent self-propagating inflammation within the pulmonary tissue that is initiated by the migration of leukocytes into the alveolar space. Recent work has demonstrated that neuronal guidance proteins are involved into the orchestration of leukocyte migration. Neogenin is a crucial guidance receptor for axonal migration, yet its role during leukocyte migration and acute inflammation is to date unknown. Here, we report that neogenin influences neutrophil migration across endothelial HMEC-1 and alveolar A549 monolayers in vitro. In vivo, Neo1(-/-) mice demonstrated 59% reduced cell count, 41% reduced TNF-α, and 76% reduced IL-6 levels within the alveolar space during lung injury. In studies employing chimeric animals, the presence of Neo1(-/-) bone marrow was associated with a 42% reduction of cell count and reduced inflammatory changes within pulmonary tissue during lung injury. The functional inhibition of neogenin through antibody injection confirmed these results and the role of neogenin for the inflammatory changes within the alveolar space. Previously unappreciated, the guidance receptor neogenin has a significant effect on the orchestration of leukocyte migration and the control of acute inflammation.     

Emerging roles for neogenin and its ligands in CNS development

It is now well established that the netrin guidance cues and their receptors comprise a major molecular guidance system driving axon pathfinding during nervous system development. One netrin receptor, neogenin, is now emerging as a key regulator of many developmental processes throughout the embryo. Unexpectedly, a new family of neogenin ligands, the repulsive guidance molecule (RGM) family, has recently been identified. The functional outcome of neogenin activation is dictated by both the nature of the ligand as well as the developmental context. Netrin-1–neogenin interactions mediate chemoattractive axon guidance, while RGMa–neogenin interactions repel axons. Neogenin is required for the establishment of the pseudostratified epithelium of the neural tube, probably by promoting cell adhesion. In addition, a role for RGMa and neogenin in neuronal differentiation has been demonstrated. While neogenin signaling cascades are poorly understood, the opposing responses of neogenin to RGMa and netrin-1 in the context of axon guidance indicates that neogenin signaling is complex and subject to tight spatiotemporal regulation. In summary, neogenin is a multifunctional receptor regulating diverse developmental processes. Thus, its contribution to neural development is proving to be considerably more extensive than originally predicted.     

Identification of the neogenin-binding site on the repulsive guidance molecule A

Repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the chick retinotectal system. RGMa, one of the 3 isoforms found in mammals, is involved in laminar patterning, cephalic neural tube closure, axon guidance, and inhibition of axonal regeneration. In addition to its roles in the nervous system, RGMa plays a role in enhancing helper T-cell activation. Binding of RGM to its receptor, neogenin, is considered necessary to transduce these signals; however, information on the binding of RGM to neogenin is limited. Using co-immunoprecipitation studies, we have identified that the RGMa region required for binding to neogenin contains amino acids (aa) 259-295. Synthesized peptide consisting of aa 284-293 directly binds to the extracellular domain (ECD) of recombinant neogenin, and addition of this peptide inhibits RGMa-induced growth cone collapse in mouse cortical neurons. Thus, we propose that this peptide is a promising lead in finding reagents capable of inhibiting RGMa signaling.     

Neogenin Regulates Skeletal Myofiber Size and Focal Adhesion Kinase and Extracellular Signal-regulated Kinase Activities In Vivo and In Vitro

A variety of signaling pathways participate in the development of skeletal muscle, but the extracellular cues that regulate such pathways in myofiber formation are not well understood. Neogenin is a receptor for ligands of the netrin and repulsive guidance molecule (RGM) families involved in axon guidance. We reported previously that neogenin promoted myotube formation by C2C12 myoblasts in vitro and that the related protein Cdo (also Cdon) was a potential neogenin coreceptor in myoblasts. We report here that mice homozygous for a gene-trap mutation in the Neo1 locus (encoding neogenin) develop myotomes normally but have small myofibers at embryonic day 18.5 and at 3 wk of age. Similarly, cultured myoblasts derived from such animals form smaller myotubes with fewer nuclei than myoblasts from control animals. These in vivo and in vitro defects are associated with low levels of the activated forms of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), both known to be involved in myotube formation, and inefficient expression of certain muscle-specific proteins. Recombinant netrin-2 activates FAK and ERK in cultured myoblasts in a neogenin- and Cdo-dependent manner, whereas recombinant RGMc displays lesser ability to activate these kinases. Together, netrin-neogenin signaling is an important extracellular cue in regulation of myogenic differentiation and myofiber size.     

Phosphatidylinositol transfer protein-alpha in netrin-1-induced PLC signalling and neurite outgrowth

Neurite extension is essential for wiring the nervous system during development. Although several factors are known to regulate neurite outgrowth, the underlying mechanisms remain unclear. Here, we provide evidence for a role of phosphatidylinositol transfer protein-alpha (PlTPalpha) in neurite extension in response to netrin-1, an extracellular guidance cue. PlTPalpha interacts with the netrin receptor DCC (deleted in colorectal cancer) and neogenin. Netrin-1 stimulates PlTPalpha binding to DCC and to phosphatidylinositol (5) phosphate [Pl(5)P], increases its lipid-transfer activity and elevates hydrolysis of phosphatidylinositol bisphosphate (PlP2). In addition, the stimulated PIP2 hydrolysis requires PlTPalpha. Furthermore, cortical explants of PlTPalpha mutant mice are defective in extending neurites in response to netrin-1. Commissural neurons from chicken embryos expressing a dominant-negative PlTPalpha mutant show reduced axon outgrowth. Morpholino-mediated knockdown of PlTPalpha expression in zebrafish embryos leads to dose-dependent defects in motor-neuron axons and reduced numbers of spinal-cord neurons. Taken together, these results identify a crucial role for PlTPalpha in netrin-1-induced neurite outgrowth, revealing a signalling mechanism for DCC/neogenin and PlTPalpha regulation.     

Connecting chronic and recurrent stress to vascular dysfunction: no relaxed role for the renin-angiotensin system

The renin-angiotensin system (RAS) is classically considered to be a protective system for volume balance and is activated during states of volume depletion. Interestingly, one of the major pathways activating the system is the sympathetic nervous system, also the primary mediator of the acute stress response. When one further examines the cells mediating the immune site of the response, which is primarily an inflammatory response leading to defense at a locally injured area, these cells all express the ANG II type 1 receptor (AGTR1). Scattered throughout the literature are reports indicating that acute and chronic stress can activate renin and increase plasma levels of components of the RAS. Moreover, there are reports describing that ANG II can modulate the distribution and function of immune cells. Since the inflammatory response is also implicated to be central in the initiation and progression of vascular damage, we propose in this review that recurrent acute stress and chronic stress can induce a state with inflammation, due to ANG II-mediated activation of inflammatory cells, specifically monocytes and lymphocytes. Such a proposal would explain a lot of the observations regarding RAS components in inflammatory cells. Despite its attractiveness, substantial research in this area would be required to substantiate this hypothesis.     

Netrin-1/neogenin interaction stabilizes multipotent progenitor cap cells during mammary gland morphogenesis

Netrin-1 and its receptors play an essential role patterning the nervous system by guiding neurons and axons to their targets. To explore whether netrin-1 organizes nonneural tissues, we examined its role in mammary gland morphogenesis. Netrin-1 is expressed in prelumenal cells, and its receptor neogenin is expressed in a complementary pattern in adjacent cap cells of terminal end buds (TEBs). We discovered that loss of either gene results in disorganized TEBs characterized by exaggerated subcapsular spaces, breaks in basal lamina, dissociated cap cells, and an increased influx of cap cells into the prelumenal compartment. Cell aggregation assays demonstrate that neogenin mediates netrin-1-dependent cell clustering. Thus, netrin-1 appears to act locally through neogenin to stabilize the multipotent progenitor (cap) cell layer during mammary gland development. Our results suggest that netrin-1 and its receptor neogenin provide an adhesive, rather than a guidance, function during nonneural organogenesis.     

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.     

Neogenin and repulsive guidance molecule signaling in the central nervous system

The repulsive guidance molecule (RGM) is a membrane-bound protein that was originally identified as an axon guidance molecule in the visual system. Functional studies have revealed that it has roles in axon guidance and laminar patterning in Xenopus and chick embryos, and in controlling cephalic neural tube closure in mouse embryos. The recent identification of neogenin as a receptor for RGM has provided evidence of the diverse functions of this ligand-receptor pair. Re-expression of RGM is observed after injury in the adult human and rat central nervous systems. Inhibition of RGM enhances growth of injured axons and promotes functional recovery after spinal cord injury in rats. Thus, re-expression of embryonic repulsive cues in adult tissues contributes to failure of axon regeneration in the central nervous system.     

Neogenin regulates neuronal survival through DAP kinase

The repulsive guidance molecule (RGM) is a membrane-bound protein that has diverse functions in the developing central nervous system. Identification of neogenin as a receptor for RGM provided evidence of its cell death-inducing activity in the absence of RGM. Here, we show that the serine/threonine kinase death-associated protein kinase (DAPK) is involved in the signal transduction of neogenin. Neogenin interacts with DAPK and reduces DAPK autophosphorylation on Ser308 in vitro. Neogenin-induced cell death is abolished in the presence of RGM or by blocking DAPK. Although neogenin overexpression or RGM downregulation in the chick neural tube in vivo induces apoptosis, coexpression of the dominant-negative mutant or small-interference RNA of DAPK attenuates this proapoptotic activity. Thus, RGM/neogenin regulates cell fate by controlling the DAPK activity.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

Aging-related hyperinflammation in endotoxemia is mediated by the α2A-adrenoceptor and CD14/TLR4 pathways

AimsSepsis is a major cause of morbidity and mortality in the elderly population. In prior studies, we have shown that in vivo, the inflammatory response in aged animals is exaggerated as compared to young animals and that this response likely accounts for the increased morbidity and mortality. Part of this uncontrolled inflammatory response in sepsis is due to the innate immune response. However, recent studies have shown that the pathogenesis of sepsis is much more complex. The adrenergic autonomic nervous system is now thought to play a key role in modulating the inflammatory response in sepsis. In this study, we hypothesize that not only is the innate immune response enhanced in response to lipopolysaccharide (LPS) in aged animals, but that the adrenergic nervous system also plays a role in the release of excess inflammatory cytokines.Main methodsMale Fischer-344 rats (young: 3 months; aged: 24 months) were used. Endotoxemia was induced by intravenous injection of lipopolysaccharide (LPS, 15 mg/kg BW). Splenic tissues were harvested and mRNA and protein were extracted. The protein expression of CD14 and TLR4, key mediators of LPS in the innate response, as well as alpha-2A adrenergic receptor (α_2A-AR) and phosphodiesterase 4D (PDE4D), as the means by which the autonomic nervous system exerts its effects were analyzed.Key findingsSplenic tissue concentrations of α_2A-AR, PDE4D, CD14, and TLR4 were significantly increased in septic aged rats as compared to aged sham rats and septic young rats. The increased expression of α_2A-AR in septic aged rats was further confirmed by immunohistochemical staining of splenic tissues.SignificanceThese data support the hypothesis that not only is the innate immune response increased in aged animals during sepsis, but that there is also an upregulated response of the adrenergic autonomic nervous system that contributes to excess proinflammatory cytokine release.     

IL-1RI (interleukin-1 receptor type I) signalling is essential for host defence and hemichannel activity during acute central nervous system bacterial infection

Staphylococcus aureus is a common aetiological agent of bacterial brain abscesses. We have previously established that a considerable IL-1 (interleukin-1) response is elicited immediately following S. aureus infection, where the cytokine can exert pleiotropic effects on glial activation and blood–brain barrier permeability. To assess the combined actions of IL-1α and IL-1β during CNS (central nervous system) infection, host defence responses were evaluated in IL-1RI (IL-1 receptor type I) KO (knockout) animals. IL-1RI KO mice were exquisitely sensitive to intracerebral S. aureus infection, as demonstrated by enhanced mortality rates and bacterial burdens within the first 24 h following pathogen exposure compared with WT (wild-type) animals. Loss of IL-1RI signalling also dampened the expression of select cytokines and chemokines, concomitant with significant reductions in neutrophil and macrophage infiltrates into the brain. In addition, the opening of astrocyte hemichannels during acute infection was shown to be dependent on IL-1RI activity. Collectively, these results demonstrate that IL-1RI signalling plays a pivotal role in the genesis of immune responses during the acute stage of brain abscess development through S. aureus containment, inflammatory mediator production, peripheral immune cell recruitment, and regulation of astrocyte hemichannel activity. Taken in the context of previous studies with MyD88 (myeloid differentiation primary response gene 88) and TLR2 (Toll-like receptor 2) KO animals, the current report advances our understanding of MyD88-dependent cascades and implicates IL-1RI signalling as a major antimicrobial effector pathway during acute brain-abscess formation.     

Repulsive guidance molecule/neogenin: a novel ligand-receptor system playing multiple roles in neural development

The repulsive guidance molecule (RGM) is a membrane-bound protein originally isolated as an axon guidance molecule in the visual system. Recently, the transmembrane protein, neogenin, has been identified as the RGM receptor. In vitro analysis with retinal explants showed that RGM repels temporal retinal axons and collapses their growth cones through neogenin-mediated signaling. However, RGM and neogenin are also broadly expressed at the early embryonic stage, suggesting that they do not only control the guidance of visual axons. Gene expression perturbation experiments in chick embryos showed that neogenin induces cell death, and its ligand, RGM, blocks the pro-apoptotic activity of neogenin. Thus, RGM/neogenin is a novel dependence ligand/receptor couple as well as an axon guidance molecular complex.     

Expression studies of neogenin and its ligand hemojuvelin in mouse tissues.

Juvenile hemochromatosis is a severe hereditary iron overload disease caused by mutations in the HJV (hemojuvelin) and HAMP (hepcidin) genes. Hepcidin is an important iron regulatory hormone, and hemojuvelin may regulate hepcidin synthesis via the multifunctional membrane receptor neogenin. We explored the expression of murine hemojuvelin and neogenin mRNAs and protein. Real-time RT-PCR analysis of 18 tissues from male and female mice was performed to examine the mRNA expression profiles. To further study protein expression and localization we used immunohistochemistry on several tissues from three mouse strains. Mouse Neo1 mRNA was detectable in the 18 tissues tested, the highest signals being evident in the ovary, uterus, and testis. Neogenin protein was observed in the brain, skeletal muscle, heart, liver, stomach, duodenum, ileum, colon, renal cortex, lung, testis, ovary, oviduct, and uterus. The spleen, thymus, and pancreas were negative for neogenin. The highest signals for Hjv mRNA were detectable in the skeletal muscle, heart, esophagus, and liver. The results indicate that Neo1 mRNA is widely expressed in both male and female mouse tissues with the highest signals detected in the reproductive system. Moreover, Hjv and Neo1 mRNAs are simultaneously expressed in skeletal muscle, heart, esophagus, and liver.     

Modulation of the acute phase response by altered expression of the IL-1 type 1 receptor or IL-1ra

A complete understanding of the role for endogenously produced interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-1 receptor antagonist (IL-1ra) in the acute phase response to inflammation remains unknown. In the present studies, knockout mice lacking either a functional IL-1 type I receptor (IL-1RI(-/-)), a TNF type I receptor (TNFR-I(-/-)), or both IL-1 type I and TNF type I receptors (IL-1RI(-/-)/TNFR-I(-/-)) received a turpentine abscess. Additional mice deficient in IL-1ra protein (IL-1ra(-/-)) or overexpressing IL-1ra protein (IL-1ra(tg)) were similarly treated. After a turpentine abscess, IL-1 receptor knockout mice exhibited an attenuated inflammatory response compared with wild-type or animals lacking a functional TNFR-I. Mice overexpressing IL-1ra also had an attenuated hepatic acute phase protein response, whereas IL-1ra knockout mice had a significantly greater hepatic acute phase response. We conclude that the inflammatory response to a turpentine abscess is the result of a balance between IL-1ra expression and IL-1 binding to its type I receptor. Endogenously produced IL-1ra plays a central role in mitigating the magnitude of the IL-1-mediated inflammatory response and, ultimately, the outcome to a turpentine abscess.     

Neogenin: one receptor, many functions

Neogenin is a multifunctional transmembrane receptor belonging to the immunoglobulin superfamily. It displays identical secondary structure to deleted in colorectal cancer (DCC), a netrin receptor that is involved in axon guidance and cell survival. Like DCC, neogenin is able to transduce signals elicited by netrin. These neogenin-netrin interactions have been implicated in tissue morphogenesis, angiogenesis, myoblast differentiation and most recently in axon guidance. Neogenin is also a receptor for repulsive guidance molecule, a glycosylphosphatidylinositol-linked protein involved in neuronal differentiation, apoptosis and repulsive axon guidance. Numerous studies have been started to elucidate the in vivo functions of neogenin, and its role in multiple aspects of development and homeostasis.     

Substance P is a key mediator of stress-induced protection from allergic sensitization via modified antigen presentation

Interaction between the nervous and immune systems greatly contributes to inflammatory disease. In organs at the interface between our body and the environment, the sensory neuropeptide substance P (SP) is one key mediator of an acute local stress response through neurogenic inflammation but may also alter cytokine balance and dendritic cell (DC) function. Using a combined murine allergic inflammation/noise stress model with C57BL/6 mice, we show in this paper that SP--released during repeated stress exposure--has the capacity to markedly attenuate inflammation. In particular, repeated stress exposure prior to allergen sensitization increases DC-nerve fiber contacts, enhances DC migration and maturation, alters cytokine balance, and increases levels of IL-2 and T regulatory cell numbers in local lymph nodes and inflamed tissue in a neurokinin 1-SP-receptor (neurokinin-1 receptor)-dependent manner. Concordantly, allergic inflammation is significantly reduced after repeated stress exposure. We conclude that SP/repeated stress prior to immune activation acts protolerogenically and thereby beneficially in inflammation.