Deleted In Colorectal Cancer Research Articles

Netrin-1 Simultaneously Suppresses Corneal Inflammation and Neovascularization

To investigate the effect of netrin-1 on alkali burn-induced corneal inflammation and neovascularization. The expression of netrin-1 and its receptors UNC5A, UNC5B, UNC5C, UNC5D, adenosine 2b receptor (A2BAR), deleted in colorectal cancer (DCC), and neogenin in normal and alkali-burned rat cornea were determined by RT-PCR and/or Western blot analysis, or immunostaining. Topical netrin-1 protein was applied to treat rat corneal alkali-burn injury for 14 consecutive days, started right after the injury or 10 days postinjury. Corneal inflammation and neovascularization were observed under slit lamp microscope. The apoptosis of corneal cells was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Corneal inflammatory cell infiltration was evaluated by immunostaining of anti-PMN and anti-ED1 antibodies. The expression of epidermal growth factor (EGF), vascular epidermal growth factor (VEGF), and pigment epithelium-derived factor (PEDF) in rat cornea was determined by Western blot analysis. Netrin-1 and its receptor UNC5B were expressed in normal rat corneal epithelium and stromal cells, and their expression decreased after corneal alkali burn. Exogenous netrin-1 administered on rat ocular surfaces resolved alkali burn-induced corneal inflammation, and also suppressed corneal neovascularization. Furthermore, netrin-1 could reverse neovascularization in alkali-burned cornea. The authors found that netrin-1 executed the functions through various mechanisms, including upregulating EGF expression, accelerating epithelial wound healing, inhibiting neutrophil and macrophage infiltration, reducing corneal cell apoptosis, and restoring the equilibrium of VEGF and PEDF in the wounded cornea. Netrin-1 could dampen inflammation, inhibit, and reverse neovascularization in alkali-burned cornea.

A genome-wide association study identifies SNP in DCC is associated with gallbladder cancer in the Japanese population

Gallbladder cancer (GC) is a relatively uncommon cancer with higher incidence in certain areas including Japan. Because of the difficulty in diagnosis, prognosis of GC is very poor. To identify genetic determinants of GC, we conducted a genome-wide association study (GWAS) in 41 GC patients and 866 controls. Association between each single-nucleotide polymorphism (SNP) with GC susceptibility was evaluated by multivariate logistic regression analysis conditioned on age and gender of subjects. SNPs that showed suggestive association (P<1 × 10(-4)) with GC were further examined in 30 cases and 898 controls. SNP rs7504990 in the DCC (deleted in colorectal cancer, 18q21.3) that encodes a netrin 1 receptor achieved a combined P-value of 7.46 × 10(-8) (OR=6.95; 95% CI=3.43-14.08). Subsequent imputation analysis identified multiple SNPs with similarly strong associations in an adjacent genomic region, where loss of heterozygosity was reported in GC and other cancers. Reduced expression of DCC was indicated to be associated with the poorly differentiated histological type, increased proliferation and metastasis through loss of adhesiveness. However, due to the limited sample size investigated here, further replication study and functional analysis would be necessary to further confirm the result of the association.

The Cbln family of proteins interact with multiple signaling pathways

Cerebellin precursor protein (Cbln1) is essential for synapse integrity in cerebellum through assembly into complexes that bridge pre-synaptic β-neurexins (Nrxn) to post-synaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1-3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, (deleted in colorectal cancer (DCC) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln null mice. Thus, Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less well-characterized role of netrin/DCC in adult brain.

Deleted in colorectal carcinoma suppresses metastasis in p53-deficient mammary tumours

Since its discovery in the early 1990s the deleted in colorectal cancer (DCC) gene, located on chromosome 18q21, has been proposed as a tumour suppressor gene as its loss is implicated in the majority of advanced colorectal and many other cancers. DCC belongs to the family of netrin 1 receptors, which function as dependence receptors as they control survival or apoptosis depending on ligand binding. However, the role of DCC as a tumour suppressor remains controversial because of the rarity of DCC-specific mutations and the presence of other tumour suppressor genes in the same chromosomal region. Here we show that in a mouse model of mammary carcinoma based on somatic inactivation of p53, additional loss of DCC promotes metastasis formation without affecting the primary tumour phenotype. Furthermore, we demonstrate that in cell cultures derived from p53-deficient mouse mammary tumours DCC expression controls netrin-1-dependent cell survival, providing a mechanistic basis for the enhanced metastatic capacity of tumour cells lacking DCC. Consistent with this idea, in vivo tumour-cell survival is enhanced by DCC loss. Together, our data support the function of DCC as a context-dependent tumour suppressor that limits survival of disseminated tumour cells.

Juvenile exposure to methylphenidate reduces cocaine reward and alters netrin-1 receptor expression in adulthood

The guidance cue netrin-1 acting on mesocorticolimbic dopamine (DA) neurons through its receptor DCC (deleted in colorectal cancer) has been implicated in the neuronal plasticity induced by psychostimulant drugs. We examined in C57/BL6 mice the effects of repeated juvenile methylphenidate (MPH) exposure on cocaine-reward sensitivity in adulthood and determined whether early MPH treatment alters adult expression of DCC in mesocorticolimbic DA regions. By using place conditioning, we show that adult mice exposed to MPH during the juvenile period are less sensitive to cocaine-reward compared to saline-controls. Furthermore, by means of immunoblotting, we demonstrate that early MPH treatment attenuates adult DCC expression in the ventral tegmental area (VTA) selectively. These results support previous evidence that developmental MPH treatment diminishes cocaine-reward in adulthood and are the first to suggest that DCC in the VTA may participate in this enduring effect.

Abstract 17337: Postconditioning with Netrin-1 Protects Against Myocardial Ischemia-Reperfusion Injury Via a DCC/ERK1/2/eNOS Mechanism

Given as a pre-conditioning mimetic, we have previously shown that netrin-1 markedly reduces myocardial ischemia-reperfusion (I/R) injury by increasing nitric oxyde (NO) bioavailability. However, its potential beneficial effect remains unknown if given as a post-conditioning agent. Langendorff perfused hearts isolated from C57BL/6 wild-type (WT) mice or DCC+/- mice underwent 20-min of global ischemia followed by 60-min of reperfusion (n=6). DCC (deleted in colorectal cancer) is one of the netrin-1 receptors we found to be however specifically involved in netrin-1 pre-conditioning induced cardioprotection. Netrin-1 was infused post-conditionally at the onset of reperfusion at 100 ng/mL concentration with and without UO126, a specific MEK1/2 inhibitor or 2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), a NO-scavenger. In WT mice, netrin-1 postconditioning significantly reduced infarct size to 17±2.5% from 43.3±4.6% in the untreated control I/R group. This phenomenon is now further confirmed using an in vivo model of myocardial ischemic injury, 30-min coronary occlusion followed by 24-hr reperfusion. UO126 or PTIO administration alone had no effect on infarct size (respectively 44.8±3% and 39.8±1.7% vs 43.3±4.6% in the untreated control I/R group) but abolished the effects of netrin-1, implicating intermediate roles of MEK/ERK1/2 and NO (respectively 44.8±2% and 43.5±2.9% vs 17±2.5% in the netrin-1 treated I/R group). Indeed, ERK1/2 and eNOS phosphorylation was significantly upregulated by netrin-1 treatment. Interestingly, the protective effect of netrin-1 was markedly diminished in the DCC+/- mice (infarct size increased to 44.5±2% from 15±2.6 % in the DCC+/+ group), indicating a dependency on DCC of netrin-1 post-conditoning induced cardioprotection. Netrin-1 also feed-forwardly upregulated DCC protein expression. Taken together, these results strongly suggest that netrin-1 protects against I/R injury in the murine heart as a post-conditioning mimetic by increasing NO production via activation of a ERK1/2/eNOS pathway, which also involves DCC activation and upregulation. These findings demonstrate that netrin-1 can serve as an innovative and powerful agent for acute treatment of myocardial infarction.

PLoss of heterozygosity (LOH) of deleted in colorectal cancer (DCC) gene and predisposition to colorectal cancer: Significant association in colorectal cancer patients of Kashmir

Ethanolic herbal extract residue of the seeds of Annona squamosa Linn. were tested against Dalton’s Lymphoma Ascites (DLA) tumour cells. Then its effect on the increased lifespan of mice with DLA tumour cells was studied. At high and low dosages, it exhibited 66.67 and 56.25% increase in the lifespan. Percentage of haemoglobin (Hb) and red blood cell (RBC) counts of treated were higher than that of tumour bearing mice. The white blood cell (WBC) counts of treated were lower than that of tumour bearing mice. This is a good index of tumour recovery. Further studies were focused on mice induced with solid tumour to record the tumour volume and lifespan. It is learnt from the UV-Vis, IR, 1H and13C NMR spectra analyses that annoglacin-B, an acetogenin, present in the extract residue of A. squamosa may be responsible for the biological activity. Key words: Dalton’s Lymphoma Ascites (DLA), red blood cell (RBC), white blood cell (WBC), haemoglobin (Hb), serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT).

Covering the Cover

Covering the Cover

A Signaling Mechanism Coupling Netrin-1/Deleted in Colorectal Cancer Chemoattraction to SNARE-Mediated Exocytosis in Axonal Growth Cones

Directed cell migration and axonal guidance are essential steps in neural development. Both processes are controlled by specific guidance cues that activate the signaling cascades that ultimately control cytoskeletal dynamics. Another essential step in migration and axonal guidance is the regulation of plasmalemma turnover and exocytosis in leading edges and growth cones. However, the cross talk mechanisms linking guidance receptors and membrane exocytosis are not understood. Netrin-1 is a chemoattractive cue required for the formation of commissural pathways. Here, we show that the Netrin-1 receptor deleted in colorectal cancer (DCC) forms a protein complex with the t-SNARE (target SNARE) protein Syntaxin-1 (Sytx1). This interaction is Netrin-1 dependent both in vitro and in vivo, and requires specific Sytx1 and DCC domains. Blockade of Sytx1 function by using botulinum toxins abolished Netrin-1-dependent chemoattraction of axons in mouse neuronal cultures. Similar loss-of-function experiments in the chicken spinal cord in vivo using dominant-negative Sytx1 constructs or RNAi led to defects in commissural axon pathfinding reminiscent to those described in Netrin-1 and DCC loss-of-function models. We also show that Netrin-1 elicits exocytosis at growth cones in a Sytx1-dependent manner. Moreover, we demonstrate that the Sytx1/DCC complex associates with the v-SNARE (vesicle SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) and that knockdown of TI-VAMP in the commissural pathway in the spinal cord results in aberrant axonal guidance phenotypes. Our data provide evidence of a new signaling mechanism that couples chemotropic Netrin-1/DCC axonal guidance and Sytx1/TI-VAMP SNARE proteins regulating membrane turnover and exocytosis.

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.

Draxin Inhibits Axonal Outgrowth through the Netrin Receptor DCC

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β–β associations found in radixin–adhesion molecule complexes. We also revealed direct interactions between the MyTH4–FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.

Draxin inhibits axonal outgrowth through the netrin receptor DCC

UNC-6 netrin, a laminin-related protein secreted from neuroglia and neurons along the ventral midline, orients migrating cells and pioneering growth cones on the nematode epidermis. UNC-5, a cell surface protein expressed on motile cells and pioneer axons, orients movements away from UNC-6 sources. UNC-40, a homolog of the cell surface proteins DCC (Deleted in Colorectal Cancer) and neogenin, is also expressed on motile cells and pioneer neurons. UNC-40 acts cell autonomously to orient movement toward UNC-6 sources. For cells coexpressing UNC-5, it helps orient movement away from UNC-6 sources. Finally, UNC-40 helps determine the dorsoventral position of cells undergoing purely longitudinal migrations. Together with the recent report that DCC is a netrin receptor in vertebrates, our results suggest that UNC-40 is a component of UNC-6 receptors on motile cells.

The activation of ezrin–radixin–moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1–induced axon outgrowth

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.

Effects of treadmill exercise on the expression of netrin-1 and its receptors in rat brain after cerebral ischemia

Recent evidence suggests that exercise improves functional outcome in animal models of cerebral ischemia. Since netrin-1 and its receptors, deleted in colorectal cancer (DCC) and uncoordinated gene 5B (Unc5B), act as important regulators in neural and vascular activities, we sought to determine whether netrin-1 and DCC and Unc5B are involved in the neuroprotective effects of exercise on rats with induced cerebral ischemia. A total of 108 rats were randomly distributed into three groups: sham-operated group (n = 12), middle cerebral artery occlusion (MCAO) group (n = 48), MCAO+treadmill exercise group (n = 48). Behavioral testing indicated that treadmill exercise could significantly improve neurologic deficits of rats with cerebral ischemia at day 14 and 28 after MCAO (n = 12, P<0.05 and P<0.01), but there was no significant difference at day 4 and 7. Quantitative reverse transcription polymerase chain reaction (qPCR) and Western blot analysis revealed that treadmill exercise enhanced netrin-1 and DCC expression, while it suppressed Unc5B expression in rat peri-ischemic brain area, especially at day 14 and 28 after MCAO (n = 4, P<0.05 or P<0.01). Immunofluorescence analysis showed that in the peri-ischemic area, netrin-1 was expressed in neuronal perikarya, DCC, however, was expressed in neural processes and peri-vascular astrocytes, while Unc5B was expressed mostly in neuronal perikarya and some processes. These results suggest that netrin-1 and its receptors DCC and Unc5B may engage in exercise-induced neural circuit remodeling in the peri-ischemic area, and exercise may promote survival of neurons in this area by regulating netrin-1-Unc5B signaling. Additionally, netrin-1 may also play a role in brain-blood barrier via DCC-immunoreactive peri-vascular astrocytes. In conclusion, we demonstrate that treadmill exercise has beneficial effects that may be attributed, at least in part, to the involvement of netrin-1 and its receptors DCC and Unc5B in the neuronal and vascular activities in brain-ischemic rats.

The Drosophila Netrin receptor frazzled/DCC functions as an invasive tumor suppressor

BackgroundLoss of heterozygosity at 18q, which includes the Deleted in Colorectal Cancer (DCC) gene, has been linked to many human cancers. However, it is unclear if loss of DCC is the specific underlying cause of these cancers. The Drosophila imaginal discs are excellent systems in which to study DCC function, as it is possible to model human tumors through the generation of somatic clones of cells bearing multiple genetic lesions. Here, these attributes of the fly system were utilized to investigate the potential tumor suppressing functions of the Drosophila DCC homologue frazzled (fra) during eye-antennal disc development.ResultsMost fra loss of function clones are eliminated during development. However, when mutant clone cells generated in the developing eye were rescued from death, partially differentiated eye cells were found outside of the normal eye field, and in extreme cases distant sites of the body. Characterization of these cells during development indicates that fra mutant cells display characteristics of invasive tumor cells, including increased levels of phospho-ERK, phospho-JNK, and Mmp-1, changes in cadherin expression, remodeling of the actin cytoskeleton, and loss of polarity. Mutation of fra promotes basement membrane degradation and invasion which are repressed by inhibition of Rho1 signaling. Although inhibition of JNK signaling blocks invasive phenotypes in some metastatic cancer models in flies, blocking JNK signaling inhibits fra mutant cell death, thereby enhancing the fra mutant phenotype.ConclusionsThe results of this investigation provide the first direct link between point mutations in fra/DCC and metastatic phenotypes in an animal model and suggest that Fra functions as an invasive tumor suppressor during Drosophila development.

A conserved juxtacrine signal regulates synaptic partner recognition in Caenorhabditis elegans

BackgroundAn essential stage of neural development involves the assembly of neural circuits via formation of inter-neuronal connections. Early steps in neural circuit formation, including cell migration, axon guidance, and the localization of synaptic components, are well described. However, upon reaching their target region, most neurites still contact many potential partners. In order to assemble functional circuits, it is critical that within this group of cells, neurons identify and form connections only with their appropriate partners, a process we call synaptic partner recognition (SPR). To understand how SPR is mediated, we previously developed a genetically encoded fluorescent trans-synaptic marker called NLG-1 GRASP, which labels synaptic contacts between individual neurons of interest in dense cellular environments in the genetic model organism Caenorhabditis elegans.ResultsHere, we describe the first use of NLG-1 GRASP technology, to identify SPR genes that function in this critical process. The NLG-1 GRASP system allows us to assess synaptogenesis between PHB sensory neurons and AVA interneurons instantly in live animals, making genetic analysis feasible. Additionally, we employ a behavioral assay to specifically test PHB sensory circuit function. Utilizing this approach, we reveal a new role for the secreted UNC-6/Netrin ligand and its transmembrane receptor UNC-40/Deleted in colorectal cancer (DCC) in SPR. Synapses between PHB and AVA are severely reduced in unc-6 and unc-40 animals despite normal axon guidance and subcellular localization of synaptic components. Additionally, behavioral defects indicate a complete disruption of PHB circuit function in unc-40 mutants. Our data indicate that UNC-40 and UNC-6 function in PHB and AVA, respectively, to specify SPR. Strikingly, overexpression of UNC-6 in postsynaptic neurons is sufficient to promote increased PHB-AVA synaptogenesis and to potentiate the behavioral response beyond wild-type levels. Furthermore, an artificially membrane-tethered UNC-6 expressed in the postsynaptic neurons promotes SPR, consistent with a short-range signal between adjacent synaptic partners.ConclusionsThese results indicate that the conserved UNC-6/Netrin-UNC-40/DCC ligand-receptor pair has a previously unknown function, acting in a juxtacrine manner to specify recognition of individual postsynaptic neurons. Furthermore, they illustrate the potential of this new approach, combining NLG-1 GRASP and behavioral analysis, in gene discovery and characterization.

Schwann cells direct peripheral nerve regeneration through the Netrin-1 receptors, DCC and Unc5H2

In the peripheral nervous system, Schwann cells (SCs) promote nerve regeneration by the secretion of trophic support molecules and the establishment of a supportive growth matrix. Elucidating factors that promote SC outgrowth following nerve injury is an important strategy for improving nerve regeneration. We identified the Netrin-1 receptors, Deleted in Colorectal Cancer (DCC) and Uncoordinated (Unc)5H2 as SC receptors that influence nerve regeneration by respectively promoting or inhibiting SC outgrowth. Significantly, we show both DCC and Unc5H2 receptors are distributed within SCs. In adult nerves, DCC is localized to the paranodes and Schmidt-Lantermann incisures of myelinating SCs, as well as along unmyelinated axons. After axotomy, DCC is prominently expressed in activated SCs at the regenerating nerve front. In contrast, Unc5H2 receptor is robustly distributed in myelinating SCs of the intact nerve and it is found at low levels in the SCs of the injury site. Local in vivo DCC siRNA mRNA knockdown at the growing tip of an injured nerve impaired SC activation and, in turn, significantly decreased axon regeneration. This forced DCC inhibition was associated with a dramatic reciprocal upregulation of Unc5H2 in the remaining SCs. Local Unc5H2 knockdown at the injury site, however, facilitated axon regrowth, indicating it has a role as an intrinsic brake to peripheral nerve regeneration. Our findings demonstrate that in adult peripheral nerves, SCs respond to DCC and Unc5H2 signaling, thereby promoting or hindering axon outgrowth and providing a novel mechanism for SC regulation during nerve regeneration.

The Netrin Receptor DCC Is Required in the Pubertal Organization of Mesocortical Dopamine Circuitry

Netrins are guidance cues involved in neural connectivity. We have shown that the netrin-1 receptor DCC (deleted in colorectal cancer) is involved in the functional organization of the mesocorticolimbic dopamine (DA) system. Adult mice with a heterozygous loss-of-function mutation in dcc exhibit changes in indexes of DA function, including DA-related behaviors. These phenotypes are only observed after puberty, a critical period in the maturation of the mesocortical DA projection. Here, we examined whether dcc heterozygous mice exhibit structural changes in medial prefrontal cortex (mPFC) DA synaptic connectivity, before and after puberty. Stereological counts of tyrosine-hydroxylase (TH)-positive varicosities were increased in the cingulate 1 and prelimbic regions of the pregenual mPFC. dcc heterozygous mice also exhibited alterations in the size, complexity, and dendritic spine density of mPFC layer V pyramidal neuron basilar dendritic arbors. Remarkably, these presynaptic and postsynaptic partner phenotypes were not observed in juvenile mice, suggesting that DCC selectively influences the extensive branching and synaptic differentiation that occurs in the maturing mPFC DA circuit at puberty. Immunolabeling experiments in wild-type mice demonstrated that DCC is segregated to TH-positive fibers innervating the nucleus accumbens, with only scarce DCC labeling in mPFC TH-positive fibers. Netrin had an inverted target expression pattern. Thus, DCC-mediated netrin-1 signaling may influence the formation/maintenance of mesocorticolimbic DA topography. In support of this, we report that dcc heterozygous mice exhibit a twofold increase in the density of mPFC DCC/TH-positive varicosities. Our results implicate DCC-mediated netrin-1 signaling in the establishment of mPFC DA circuitry during puberty.

Structural basis of cargo recognition by the myosin-X MyTH4-FERM domain

Myosin-X is an important unconventional myosin that is critical for cargo transportation to filopodia tips and is also utilized in spindle assembly by interacting with microtubules. We present a series of structural and biochemical studies of the myosin-X tail domain cassette, consisting of myosin tail homology 4 (MyTH4) and FERM domains in complex with its specific cargo, a netrin receptor DCC (deleted in colorectal cancer). The MyTH4 domain is folded into a helical VHS-like structure and is associated with the FERM domain. We found an unexpected binding mode of the DCC peptide to the subdomain C groove of the FERM domain, which is distinct from previously reported β-β associations found in radixin-adhesion molecule complexes. We also revealed direct interactions between the MyTH4-FERM cassette and tubulin C-terminal acidic tails, and identified a positively charged patch of the MyTH4 domain, which is involved in tubulin binding. We demonstrated that both DCC and integrin bindings interfere with microtubule binding and that DCC binding interferes with integrin binding. Our results provide the molecular basis by which myosin-X facilitates alternative dual binding to cargos and microtubules.