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

Retinoid X receptor gamma signaling accelerates CNS remyelination

The molecular basis of CNS myelin regeneration (remyelination) is poorly understood. We generated a comprehensive transcriptional profile of the separate stages of spontaneous remyelination that follow focal demyelination in the rat CNS and found that transcripts that encode the retinoid acid receptor RXR-γ were differentially expressed during remyelination. Cells of the oligodendrocyte lineage expressed RXR-γ in rat tissues that were undergoing remyelination and in active and remyelinated multiple sclerosis lesions. Knockdown of RXR-γ by RNA interference or RXR-specific antagonists severely inhibited oligodendrocyte differentiation in culture. In mice that lacked RXR-γ, adult oligodendrocyte precursor cells efficiently repopulated lesions after demyelination, but showed delayed differentiation into mature oligodendrocytes. Administration of the RXR agonist 9-cis-retinoic acid to demyelinated cerebellar slice cultures and to aged rats after demyelination caused an increase in remyelinated axons. Our results indicate that RXR-γ is a positive regulator of endogenous oligodendrocyte precursor cell differentiation and remyelination and might be a pharmacological target for regenerative therapy in the CNS

The Polarity Protein Scribble Regulates Myelination and Remyelination in the Central Nervous System

The development and regeneration of myelin by oligodendrocytes, the myelin-forming cells of the central nervous system (CNS), requires profound changes in cell shape that lead to myelin sheath initiation and formation. Here, we demonstrate a requirement for the basal polarity complex protein Scribble in CNS myelination and remyelination. Scribble is expressed throughout oligodendroglial development and is up-regulated in mature oligodendrocytes where it is localised to both developing and mature CNS myelin sheaths. Knockdown of Scribble expression in cultured oligodendroglia results in disrupted morphology and myelination initiation. When Scribble expression is conditionally eliminated in the myelinating glia of transgenic mice, myelin initiation in CNS is disrupted, both during development and following focal demyelination, and longitudinal extension of the myelin sheath is disrupted. At later stages of myelination, Scribble acts to negatively regulate myelin thickness whilst suppressing the extracellular signal-related kinase (ERK)/mitogen-activated protein kinase (MAP) kinase pathway, and localises to non-compact myelin flanking the node of Ranvier where it is required for paranodal axo-glial adhesion. These findings demonstrate an essential role for the evolutionarily-conserved regulators of intracellular polarity in myelination and remyelination

The Precision Interventions for Severe and/or Exacerbation-Prone (PrecISE) Asthma Network: an overview of Network organization, procedures and interventions

Asthma is a heterogeneous disease, with multiple underlying inflammatory pathways and structural airway abnormalities that impact disease persistence and severity. Recent progress has been made in developing targeted asthma therapeutics, especially for subjects with eosinophilic asthma. However, there is an unmet need for new approaches to treat patients with severe and exacerbation prone asthma, who contribute disproportionately to disease burden. Extensive deep phenotyping has revealed the heterogeneous nature of severe asthma and identified distinct disease subtypes. A current challenge in the field is to translate new and emerging knowledge about different pathobiologic mechanisms in asthma into patient-specific therapies, with the ultimate goal of modifying the natural history of disease. Here we describe the Precision Interventions for Severe and/or Exacerbation Prone Asthma (PrecISE) Network, a groundbreaking collaborative effort of asthma researchers and biostatisticians from around the U.S. The PrecISE Network was designed to conduct phase II/proof of concept clinical trials of precision interventions in the severe asthma population, and is supported by the National Heart Lung and Blood Institute of the National Institutes of Health. Using an innovative adaptive platform trial design, the Network will evaluate up to six interventions simultaneously in biomarker-defined subgroups of subjects. We review the development and organizational structure of the Network, and choice of interventions being studied. We hope that the PrecISE Network will enhance our understanding of asthma subtypes and accelerate the development of therapeutics for of severe asthma

Netrin and netrin receptor function in glial motility and myelination

Netrin-1 and its receptors play crucial roles during embryogenesis, guiding axon and neuronal cell migration. Here, roles of netrins and their receptors in glial function were investigated. In the embryonic spinal cord, netrin-1 expressed at the ventral midline orients axon extension. Spinal oligodendrocyte precursor (OP) cells are born close to, and migrate away from, the ventral midline. We find that OPs express DCC and UNC5 netrin receptors and, in an in vitro microchemotaxis assay, are repelled by a netrin-1 gradient. In the absence of netrin-1 or DCC function in vivo, fewer OPs migrate from the ventral to the dorsal embryonic spinal cord, consistent with netrin-1 acting as a repellent guidance cue for these cells.In the adult CNS, oligodendrocytes continue to express DCC and UNC5 receptors, and upregulate netrin-1 expression. Our findings indicate that netrin-1 and its receptors are localized to paranodal axo-glial junctions, specialized cell-cell adhesions between non-compact myelin loops and axons. In myelinating cerebellar slice cultures derived from neonatal DCC-/- and netrin-1-/- mice, paranodes develop and mature normally but later become disorganized, resulting in loss of domain segregation at the nodal region. These data suggest that netrin-1 and DCC are essential for the maintenance of paranodal junctions, and may be indicative of a wider role in mediating cell-cell contacts in the adult.Netrin-1, DCC, and UNC5 homologues have also been identified as putative tumor suppressors, and their expression is downregulated in many cancers, including glial tumors. In our studies, netrins were found to act as autocrine factors that restrain human glioblastoma cell migration, slowing cell movement and inhibiting the formation of focal contacts associated with lamellipodial protrusion and membrane extension. DCC and UNC5 homologues have previously been proposed to inhibit tumorigenesis by inducing cell death when unbound by netrin. However, we found no evidence of increased cell death in the absence of netrin function in oligodendrocyte precursors, oligodendrocytes or glioma cells. Instead, we find that netrins act as long-range guidance cues during glial precursor migration during development, while acting at short distances to stabilize cell-cell and cell-matrix interactions of mature glia and glial tumor cells, maintaining tissue organization and preventing inappropriate cell motility

In vitro modeling of central nervous system myelination and remyelination

This review aims to summarize the current techniques to study myelination and remyelination in culture systems. We attempt to put these into historical context, and to identify the strengths and weaknesses of each approach, which vary depending on the experimental question to be tested. We discuss the difficulty and importance of quantification of myelination and in particular remyelination. Finally, we provide our predictions of how these techniques will and should develop in the future

Netrin-1 is a chemoattractant for DCC-expressing glioblastoma cells.

<p>(A,B) Addition of 100 ng/ml netrin-1 to the bottom compartment (NB) of the transfilter microchemotaxis assay significantly increased U87 cell migration compared to control (medium alone). NB: netrin-1 bottom; NTB: netrin-1 top and bottom; NB DCC_FB: netrin-1 bottom, DCC function-blocking antibody top and bottom. Similar results were obtained in assays lasting (A) 16 hours and (B) 48 hours. (C) Netrin-1 in the bottom compartment had no effect on the migration of U343 or U373 cells. (D) U343 cells transfected with a DCC expression construct (U343D control) reduced their rate of migration relative to the parental line (U343P). Increased migration of DCC-transfected U343 cells was evoked by netrin-1 in the bottom compartment (U343D NB), but not uniform netrin-1 (NTB). DCC_FB blocked this response (U343D DCC_FB). (F) Transfection of U373 cells with DCC produced responses similar to U343 cells, which mimic those seen in DCC expressing U87 cells. Number of cells migrated is per 10X objective field. 16 hr assays in all panels except (B). * p<0.05 vs. control (A,B), U343P (D) or U373P (E). § p<0.05 vs. U343D control (E) or U373D control (F).</p