Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands. Here, we show that signal integration occurs through the formation of a ternary NEO1-NET1-RGM complex, which triggers reciprocal silencing of downstream signaling. Our NEO1-NET1-RGM structures reveal a "trimer-of-trimers" super-assembly, which exists in the cell membrane. Super-assembly formation results in inhibition of RGMA-NEO1-mediated growth cone collapse and RGMA- or NET1-NEO1-mediated neuron migration, by preventing formation of signaling-compatible RGM-NEO1 complexes and NET1-induced NEO1 ectodomain clustering. These results illustrate how simultaneous binding of ligands with opposing functions, to a single receptor, does not lead to competition for binding, but to formation of a super-complex that diminishes their functional outputs.

Structural Perspectives on Axon Guidance.

Axon guidance relies on a combinatorial code of receptor and ligand interactions that direct adhesive/attractive and repulsive cellular responses. Recent structural data have revealed many of the molecular mechanisms that govern these interactions and enabled the design of sophisticated mutant tools to dissect their biological functions. Here, we discuss the structure/function relationships of four major classes of guidance cues (ephrins, semaphorins, slits, netrins) and examples of morphogens (Wnt, Shh) and of cell adhesion molecules (FLRT). These cell signaling systems rely on specific modes of receptor-ligand binding that are determined by selective binding sites; however, defined structure-encoded receptor promiscuity also enables cross talk between different receptor/ligand families and can also involve extracellular matrix components. A picture emerges in which a multitude of highly context-dependent structural assemblies determines the finely tuned cellular behavior required for nervous system development.

Oncogenic stress-induced Netrin is a humoral signaling molecule that reprograms systemic metabolism in Drosophila.

Cancer exerts pleiotropic, systemic effects on organisms, leading to health deterioration and eventually to organismal death. How cancer induces systemic effects on remote organs and the organism itself still remains elusive. Here we describe a role for NetrinB (NetB), a protein with a particularly well-characterized role as a tissue-level axon guidance cue, in mediating oncogenic stress-induced organismal, metabolic reprogramming as a systemic humoral factor. In Drosophila, Ras-induced dysplastic cells upregulate and secrete NetB. Inhibition of either NetB from the transformed tissue or its receptor in the fat body suppresses oncogenic stress-induced organismal death. NetB from the dysplastic tissue remotely suppresses carnitine biosynthesis in the fat body, which is critical for acetyl-CoA generation and systemic metabolism. Supplementation of carnitine or acetyl-CoA ameliorates organismal health under oncogenic stress. This is the first identification, to our knowledge, of a role for the Netrin molecule, which has been studied extensively for its role within tissues, in humorally mediating systemic effects of local oncogenic stress on remote organs and organismal metabolism.

The role of microtubule-associated protein tau in netrin-1 attractive signaling.

Direct binding of netrin receptors with dynamic microtubules (MTs) in the neuronal growth cone plays an important role in netrin-mediated axon guidance. However, how netrin-1 (NTN1) regulates MT dynamics in axon turning remains a major unanswered question. Here, we show that the coupling of netrin-1 receptor DCC with tau (MAPT)-regulated MTs is involved in netrin-1-promoted axon attraction. Tau directly interacts with DCC and partially overlaps with DCC in the growth cone of primary neurons. Netrin-1 induces this interaction and the colocalization of DCC and tau in the growth cone. The netrin-1-induced interaction of tau with DCC relies on MT dynamics and TUBB3, a highly dynamic ß-tubulin isotype in developing neurons. Netrin-1 increased cosedimentation of DCC with tau and TUBB3 in MTs, and knockdown of either tau or TUBB3 mutually blocked this effect. Downregulation of endogenous tau levels by tau shRNAs inhibited netrin-1-induced axon outgrowth, branching and commissural axon attraction in vitro, and led to defects in spinal commissural axon projection in vivo. These findings suggest that tau is a key MT-associated protein coupling DCC with MT dynamics in netrin-1-promoted axon attraction.

Netrin 1 directs vascular patterning and maturity in the developing kidney.

The intricate vascular system of the kidneys supports body fluid and organ homeostasis. However, little is known about how vascular architecture is established during kidney development. More specifically, how signals from the kidney influence vessel maturity and patterning remains poorly understood. Netrin 1 (Ntn1) is a secreted ligand that is crucial for vessel and neuronal guidance. Here, we demonstrate that Ntn1 is expressed by Foxd1+ stromal progenitors in the developing mouse kidney and conditional deletion (Foxd1GC/+;Ntn1fl/fl) results in hypoplastic kidneys with extended nephrogenesis. Wholemount 3D analyses additionally revealed the loss of a predictable vascular pattern in Foxd1GC/+;Ntn1fl/fl kidneys. As vascular patterning has been linked to vessel maturity, we investigated arterialization. Quantification of the CD31+ endothelium at E15.5 revealed no differences in metrics such as the number of branches or branch points, whereas the arterial vascular smooth muscle metrics were significantly reduced at both E15.5 and P0. In support of our observed phenotypes, whole kidney RNA-seq revealed disruptions to genes and programs associated with stromal cells, vasculature and differentiating nephrons. Together, our findings highlight the significance of Ntn1 to proper vascularization and kidney development.

Stromal netrin 1 coordinates renal arteriogenesis and mural cell differentiation.

The kidney vasculature has a complex architecture that is essential for renal function. The molecular mechanisms that direct development of kidney blood vessels are poorly characterized. We identified a regionally restricted, stroma-derived signaling molecule, netrin 1 (Ntn1), as a regulator of renal vascular patterning in mice. Stromal progenitor (SP)-specific ablation of Ntn1 (Ntn1SPKO) resulted in smaller kidneys with fewer glomeruli, as well as profound defects of the renal artery and transient blood flow disruption. Notably, Ntn1 ablation resulted in loss of arterial vascular smooth muscle cell (vSMC) coverage and in ectopic SMC deposition at the kidney surface. This was accompanied by dramatic reduction of arterial tree branching that perdured postnatally. Transcriptomic analysis of Ntn1SPKO kidneys revealed dysregulation of vSMC differentiation, including downregulation of Klf4, which we find expressed in a subset of SPs. Stromal Klf4 deletion similarly resulted in decreased smooth muscle coverage and arterial branching without, however, the disruption of renal artery patterning and perfusion seen in Ntn1SPKO. These data suggest a stromal Ntn1-Klf4 axis that regulates stromal differentiation and reinforces stromal-derived smooth muscle as a key regulator of renal blood vessel formation.

Systematic analysis of the Frazzled receptor interactome establishes previously unreported regulators of axon guidance.

The Netrin receptor Dcc and its Drosophila homolog Frazzled play crucial roles in diverse developmental process, including axon guidance. In Drosophila, Fra regulates midline axon guidance through a Netrin-dependent and a Netrin-independent pathway. However, what molecules regulate these distinct signaling pathways remain unclear. To identify Fra-interacting proteins, we performed affinity purification mass spectrometry to establish a neuronal-specific Fra interactome. In addition to known interactors of Fra and Dcc, including Netrin and Robo1, our screen identified 85 candidate proteins, the majority of which are conserved in humans. Many of these proteins are expressed in the ventral nerve cord, and gene ontology, pathway analysis and biochemical validation identified several previously unreported pathways, including the receptor tyrosine phosphatase Lar, subunits of the COP9 signalosome and Rho-5, a regulator of the metalloprotease Tace. Finally, genetic analysis demonstrates that these genes regulate axon guidance and may define as yet unknown signaling mechanisms for Fra and its vertebrate homolog Dcc. Thus, the Fra interactome represents a resource to guide future functional studies.

TOM-1/tomosyn acts with the UNC-6/netrin receptor UNC-5 to inhibit growth cone protrusion in Caenorhabditis elegans.

In the polarity/protrusion model of growth cone repulsion from UNC-6/netrin, UNC-6 first polarizes the growth cone of the VD motor neuron axon via the UNC-5 receptor, and then regulates protrusion asymmetrically across the growth cone based on this polarity. UNC-6 stimulates protrusion dorsally through the UNC-40/DCC receptor, and inhibits protrusion ventrally through UNC-5, resulting in net dorsal growth. Previous studies showed that UNC-5 inhibits growth cone protrusion via the flavin monooxygenases and potential destabilization of F-actin, and via UNC-33/CRMP and restriction of microtubule plus-end entry into the growth cone. We show that UNC-5 inhibits protrusion through a third mechanism involving TOM-1/tomosyn. A short isoform of TOM-1 inhibited protrusion downstream of UNC-5, and a long isoform had a pro-protrusive role. TOM-1/tomosyn inhibits formation of the SNARE complex. We show that UNC-64/syntaxin is required for growth cone protrusion, consistent with a role of TOM-1 in inhibiting vesicle fusion. Our results are consistent with a model whereby UNC-5 utilizes TOM-1 to inhibit vesicle fusion, resulting in inhibited growth cone protrusion, possibly by preventing the growth cone plasma membrane addition required for protrusion.

Netrin-1 and its receptor DCC modulate survival and death of dopamine neurons and Parkinson's disease features.

The netrin-1/DCC ligand/receptor pair has key roles in central nervous system (CNS) development, mediating axonal, and neuronal navigation. Although expression of netrin-1 and DCC is maintained in the adult brain, little is known about their role in mature neurons. Notably, netrin-1 is highly expressed in the adult substantia nigra, leading us to investigate a role of the netrin-1/DCC pair in adult nigral neuron fate. Here, we show that silencing netrin-1 in the adult substantia nigra of mice induces DCC cleavage and a significant loss of dopamine neurons, resulting in motor deficits. Because loss of adult dopamine neurons and motor impairments are features of Parkinson's disease (PD), we studied the potential impact of netrin-1 in different animal models of PD. We demonstrate that both overexpression of netrin-1 and brain administration of recombinant netrin-1 are neuroprotective and neurorestorative in mouse and rat models of PD. Of interest, we observed that netrin-1 levels are significantly reduced in PD patient brain samples. These results highlight the key role of netrin-1 in adult dopamine neuron fate, and the therapeutic potential of targeting netrin-1 signaling in PD.

A role for axon-glial interactions and Netrin-G1 signaling in the formation of low-threshold mechanoreceptor end organs.

Low-threshold mechanoreceptors (LTMRs) and their cutaneous end organs convert light mechanical forces acting on the skin into electrical signals that propagate to the central nervous system. In mouse hairy skin, hair follicle-associated longitudinal lanceolate complexes, which are end organs comprising LTMR axonal endings that intimately associate with terminal Schwann cell (TSC) processes, mediate LTMR responses to hair deflection and skin indentation. Here, we characterized developmental steps leading to the formation of Aß rapidly adapting (RA)-LTMR and Aδ-LTMR lanceolate complexes. During early postnatal development, Aß RA-LTMRs and Aδ-LTMRs extend and prune cutaneous axonal branches in close association with nascent TSC processes. Netrin-G1 is expressed in these developing Aß RA-LTMR and Aδ-LTMR lanceolate endings, and Ntng1 ablation experiments indicate that Netrin-G1 functions in sensory neurons to promote lanceolate ending elaboration around hair follicles. The Netrin-G ligand (NGL-1), encoded by Lrrc4c, is expressed in TSCs, and ablation of Lrrc4c partially phenocopied the lanceolate complex deficits observed in Ntng1 mutants. Moreover, NGL-1-Netrin-G1 signaling is a general mediator of LTMR end organ formation across diverse tissue types demonstrated by the fact that Aß RA-LTMR endings associated with Meissner corpuscles and Pacinian corpuscles are also compromised in the Ntng1 and Lrrc4c mutant mice. Thus, axon-glia interactions, mediated in part by NGL-1-Netrin-G1 signaling, promote LTMR end organ formation.

Increased Netrin downstream of overactive Hedgehog signaling disrupts optic fissure formation.

BACKGROUND: Uveal coloboma, a developmental eye defect, is caused by failed development of the optic fissure, a ventral structure in the optic stalk and cup where axons exit the eye and vasculature enters. The Hedgehog (Hh) signaling pathway regulates optic fissure development: loss-of-function mutations in the Hh receptor ptch2 produce overactive Hh signaling and can result in coloboma. We previously proposed a model where overactive Hh signaling disrupts optic fissure formation by upregulating transcriptional targets acting both cell- and non-cell-autonomously. Here, we examine the Netrin family of secreted ligands as candidate Hh target genes. RESULTS: We find multiple Netrin ligands upregulated in the zebrafish ptch2 mutant during optic fissure development. Using a gain-of-function approach to overexpress Netrin in a spatiotemporally specific manner, we find that netrin1a or netrin1b overexpression is sufficient to cause coloboma and disrupt wild-type optic fissure formation. We used loss-of-function alleles, CRISPR/Cas9 mutagenesis, and morpholino knockdown to test if loss of Netrin can rescue coloboma in the ptch2 mutant: loss of netrin genes does not rescue the ptch2 mutant phenotype. CONCLUSION: These results suggest that Netrin is sufficient but not required to disrupt optic fissure formation downstream of overactive Hh signaling in the ptch2 mutant.

Membrane trafficking of synaptic adhesion molecules.

Synapse formation and stabilization are aided by several families of adhesion molecules, which are generally seen as specialized surface receptors. The function of most surface receptors, including adhesion molecules, is modulated in non-neuronal cells by the processes of endocytosis and recycling, which control the number of active receptors found on the cell surface. These processes have not been investigated extensively at the synapse. This review focuses on the current status of this topic, summarizing general findings on the membrane trafficking of the most prominent synaptic adhesion molecules. Remarkably, evidence for endocytosis processes has been obtained for many synaptic adhesion proteins, including dystroglycans, latrophilins, calsyntenins, netrins, teneurins, neurexins, neuroligins and neuronal pentraxins. Less evidence has been obtained on their recycling, possibly because of the lack of specific assays. We conclude that the trafficking of the synaptic adhesion molecules is an important topic, which should receive more attention in the future.

Frazzled/Dcc acts independently of Netrin to promote germline survival during Drosophila oogenesis.

The Netrin receptor Frazzled/Dcc (Fra in Drosophila) functions in diverse tissue contexts to regulate cell migration, axon guidance and cell survival. Fra signals in response to Netrin to regulate the cytoskeleton and also acts independently of Netrin to directly regulate transcription during axon guidance in Drosophila. In other contexts, Dcc acts as a tumor suppressor by directly promoting apoptosis. In this study, we report that Fra is required in the Drosophila female germline for the progression of egg chambers through mid-oogenesis. Loss of Fra in the germline, but not the somatic cells of the ovary, results in the degeneration of egg chambers. Although a failure in nutrient sensing and disruptions in egg chamber polarity can result in degeneration at mid-oogenesis, these factors do not appear to be affected in fra germline mutants. However, similar to the degeneration that occurs in those contexts, the cell death effector Dcp-1 is activated in fra germline mutants. The function of Fra in the female germline is independent of Netrin and requires the transcriptional activation domain of Fra. In contrast to the role of Dcc in promoting cell death, our observations reveal a role for Fra in regulating germline survival by inhibiting apoptosis.

IGF2BP2 inhibits invasion and migration of clear cell renal cell carcinoma via targeting Netrin-4 in an m6A-dependent manner.

Clear cell renal cell carcinoma (ccRCC), the most common subtype of renal cell carcinoma, often leads to a poor prognosis due to metastasis. The investigation of N6-methyladenosine (m6A) methylation, a crucial RNA modification, and its role in ccRCC, particularly through the m6A reader insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2), revealed significant insights. We found that IGF2BP2 was notably downregulated in ccRCC, which correlated with tumor aggressiveness and poor prognosis. Thus, IGFBP2 has emerged as an independent prognostic factor of ccRCC. Moreover, a strong positive correlation was observed between the expression of IGF2BP2 and Netrin-4. Netrin-4 was also downregulated in ccRCC, and its lower levels were associated with increased malignancy and poor prognosis. Overexpression of IGF2BP2 and Netrin-4 suppressed the invasion and migration of ccRCC cells, while Netrin-4 knockdown reversed these effects in ccRCC cell lines. RNA immunoprecipitation (RIP)-quantitative polymerase chain reaction validated the robust enrichment of Netrin-4 mRNA in anti-IGF2BP2 antibody immunoprecipitates. MeRlP showed significantly increased Netrin4 m6A levels after lGF2BP2 overexpression. Moreover, we found that IGF2BP2 recognized and bound to the m6A site within the coding sequence of Netrin-4, enhancing its mRNA stability. Collectively, these results showed that IGF2BP2 plays a suppressive role in the invasion and migration of ccRCC cells by targeting Netrin-4 in an m6A-dependent manner. These findings underscore the potential of IGF2BP2/Netrin-4 as a promising prognostic biomarker and therapeutic target in patients with ccRCC metastasis.

Human Retinal Ganglion Cells Respond to Evolutionarily Conserved Chemotropic Cues for Intra Retinal Guidance and Regeneration.

Retinal ganglion cells (RGCs) connect the retina with the higher centers in the brain for visual perception. Their degeneration leads to irreversible vision loss in patients with glaucoma. The mechanism underlying human RGCs (hRGCs) axon growth and guidance remains poorly understood because hRGCs are born during development and connections with the central targets are established before birth. Here, using RGCs directly generated from human embryonic stem cells, we demonstrate that hRGCs express a battery of guidance receptors. These receptors allow hRGCs to read the spatially arrayed chemotropic cues in the developing rat retina for the centripetal orientation of axons toward the optic disc, suggesting that the mechanism of intraretinal guidance is conserved in hRGCs. The centripetal orientation of hRGCs axons is not only in response to chemorepulsion but also involves chemoattraction, mediated by Netrin-1/DCC interaction. The spatially arrayed chemotropic cues differentially influence hRGCs physiological responses, suggesting that neural activity of hRGCs and axon growth may be coupled during inter-retinal guidance. In addition, we demonstrate that Netrin-1/DCC interaction, besides promoting axon growth, facilitates hRGCs axon regeneration by recruiting the mTOR signaling pathway. The diverse influence of Netrin-1/DCC interaction ranging from axon growth to regeneration may involve recruitment of multiple intracellular signaling pathways as revealed by transcriptome analysis of hRGCs. From the perspective of ex vivo stem cell approach to glaucomatous degeneration, our findings posit that ex vivo generated hRGCs can read the intraretinal cues for guidance toward the optic disc, the first step required for connecting with the central target to restore vision.

Netrin-1 attenuates cerebral ischemia/reperfusion injury by limiting mitochondrial ROS and Ca2+ levels via activation of AKT phosphorylation and mitochondrial m-AAA protease AFG3L2.

Cerebral ischemia-reperfusion (I/R) injury as the consequence of revascularization after ischemic stroke is associated with mitochondrial dysfunction, oxidative stress, and neuron loss. In this study, we used a deprivation/reoxygenation (OGD/R) model to determine whether interactions between Netrin-1, AKT, and the mitochondrial AAA protease AFG3L2 could influence mitochondrial function in neurons after I/R. We found that Netrin-1 protects primary cortical neurons from OGD/R-induced cell death and regulates mitochondrial reactive oxygen species (ROS) and Ca2+ levels. The accumulation of mitochondrial calcium uniporter (MCU) subunits was monitored in cells by immunoblot analysis. Although the regulatory subunits MICU1 and MICU2 were relatively unaffected, the accumulation of the essential MCU regulator (EMRE) subunit was impaired. In OGD/R-induced cells, the 7 kDa form of EMRE was significantly reduced. Netrin-1 inhibited the accumulation of EMRE and mitochondrial Ca2+ levels by upregulating AFG3L2 and AKT activation. Loss of AFG3L2 or inhibition of AKT increased levels of 7 kDa EMRE. Moreover, overexpression of AKT increased the expression of AFG3L2 in Netrin-1-knockdown neurons after OGD/R. Our results demonstrate that Netrin-1 enhanced AFG3L2 protein expression via activation of AKT. We also observed that overexpression of Netrin-1 significantly reduced infarction size in an I/R-induced brain injury model in rats but not when AKT was inhibited. Our data suggest that AFG3L2 is a protein substrate of AKT and indicate that Netrin-1 attenuates cerebral I/R injury by limiting mitochondrial ROS and Ca2+ levels through activating AKT phosphorylation and AFG3L2.

Netrin-1-CD146 and netrin-1-S100A9 are associated with early stage of lymph node metastasis in colorectal cancer.

BACKGROUND: The netrin-1/CD146 pathway regulates colorectal cancer (CRC) liver metastasis, angiogenesis, and vascular development. However, few investigations have yet examined the biological function of netrin-1/CD146 complex in CRC. In this work, we investigated the relationship between the netrin-1/CD146 axis and S100 proteins in sentinel lymph node, and revealed a possible new clue for vascular metastasis of CRC. METHODS: The expression levels of netrin-1 and CD146 proteins in CRC, as well as S100A8 and S100A9 proteins in the sentinel lymph nodes were determined by immunohistochemistry. Using GEPIA and UALCAN, we analyzed netrin-1 and CD146 gene expression in CRC, their association with CRC stage, and their expression levels and prognosis in CRC patients. RESULTS: The expression level of netrin-1 in N1a+1b (CRC lymphatic metastasis groups, exculded N1c) was positively increased with N0 (p = 0.012). The level of netrin-1 protein was positively correlated with CD146 protein (p < 0.05). The level of S100A9 protein was positively correlated with CD146 protein (r = 0.492, p = 0.007). Moreover, netrin-1 expression was obviously correlated with S100A9 expression in the N1 stage (r = 0.867, p = 0.000). CD146 level was correlated with S100A9 level in the N2 stage (r = 0.731, p = 0.039). CD146 mRNA expression was higher in normal colorectal tissues than in CRC (p < 0.05). Netrin-1 and CD146 expression were not significantly associated with the tumor stages and prognosis of patients with CRC (p > 0.05). CONCLUSIONS: The netrin-1/CD146 and netrin-1/S100A9 axis in CRC tissues might related with early stage of lymph node metastasis, thus providing potential novel channels for blocking lymphatic metastasis and guiding biomarker discovery in CRC patients.

Netrin-1 protects blood-brain barrier (BBB) integrity after cerebral ischemia-reperfusion by activating the Kruppel-like factor 2 (KLF2)/occludin pathway.

Ischemia/reperfusion (I/R)-induced neural damage and neuroinflammation have been associated with pathological progression during stroke. Netrin-1 is an important member of the family of laminin-related secreted proteins, which plays an important role in governing axon elongation. However, it is unknown whether Netrin-1 possesses a beneficial role in stroke. Here, we employed the middle cerebral artery occlusion (MCAO) model to study the function of Netrin-1 in alleviating brain injuries. Our results demonstrate that Netrin-1 rescued poststroke neurological deficits and inhibited production of the inflammatory cytokines such as interleukin 6 (IL-6) and endothelial chemokine (C-X-C motif) ligand 1 (Cxcl1). Importantly, Netrin-1 protected against MCAO-induced dysfunction of the blood-brain barrier (BBB) in mice and a reduction in the expression of the tight junction (TJ) protein occludin. Additionally, we report that Netrin-1 could ameliorate oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury and prevent aggravation in endothelial monolayer permeability in bEnd.3 human brain microvascular endothelial cells (HBMVECs). Mechanistically, Netrin-1 ameliorated OGD/R-induced decrease in occludin and Kruppel-like factor 2 (KLF2) in HBMVECs. Notably, silencing of KLF2 abolished the beneficial effects of Netrin-1 in protecting endothelial permeability and occludin expression, suggesting that these effects are mediated by KLF2. In conclusion, our findings suggest that Netrin-1 could constitute a novel therapeutic strategy for ischemic stroke.

Netrin-4 synthesized in satellite cell-derived myoblasts stimulates autonomous fusion.

Satellite cells are indispensable for skeletal muscle regeneration and hypertrophy by forming nascent myofibers (myotubes). They synthesize multi-potent modulator netrins (secreted subtypes: netrin-1, -3, and -4), originally found as classical neural axon guidance molecules. While netrin-1 and -3 have key roles in myogenic differentiation, the physiological significance of netrin-4 is still unclear. This study examined whether netrin-4 regulates myofiber type commitment and myotube formation. Initially, the expression profiles indicated that satellite cells isolated from the extensor digitorum longus muscle (EDL muscle: fast-twitch myofiber-abundant) expressed slightly more netrin-4 than the soleus muscle (slow-type abundant) cells. As netrin-4 knockdown inhibited both slow- and fast-type myotube formation, netrin-4 may not directly regulate myofiber type commitment. However, netrin-4 knockdown in satellite cell-derived myoblasts reduced the myotube fusion index, while exogenous netrin-4 promoted myotube formation, even though netrin-4 expression level was maximum during the initiation stage of myogenic differentiation. Furthermore, netrin-4 knockdown also inhibited MyoD (a master transcriptional factor of myogenesis) and Myomixer (a myoblast fusogenic molecule) expression. These data suggest that satellite cells synthesize netrin-4 during myogenic differentiation initiation to promote their own fusion, stimulating the MyoD-Myomixer signaling axis.

Δ40p53 isoform up-regulates netrin-1/UNC5B expression and potentiates netrin-1 pro-oncogenic activity.

Netrin-1, a secreted protein recently characterized as a relevant cancer therapeutic target, is the antiapoptotic ligand of the dependence receptors deleted in colorectal carcinoma and members of the UNC5H family. Netrin-1 is overexpressed in several aggressive cancers where it promotes cancer progression by inhibiting cell death induced by its receptors. Interference of its binding to its receptors has been shown, through the development of a monoclonal neutralizing antinetrin-1 antibody (currently in phase II of clinical trial), to actively induce apoptosis and tumor growth inhibition. The transcription factor p53 was shown to positively regulate netrin-1 gene expression. We show here that netrin-1 could be a target gene of the N-terminal p53 isoform Δ40p53, independent of full-length p53 activity. Using stable cell lines, harboring wild-type or null-p53, in which Δ40p53 expression could be finely tuned, we prove that Δ40p53 binds to and activates the netrin-1 promoter. In addition, we show that forcing immortalized human skeletal myoblasts to produce the Δ40p53 isoform, instead of full-length p53, leads to the up-regulation of netrin-1 and its receptor UNC5B and promotes cell survival. Indeed, we demonstrate that netrin-1 interference, in the presence of Δ40p53, triggers apoptosis in cancer and primary cells, leading to tumor growth inhibition in preclinical in vivo models. Finally, we show a positive correlation between netrin-1 and Δ40p53 gene expression in human melanoma and colorectal cancer biopsies. Hence, we propose that inhibition of netrin-1 binding to its receptors should be a promising therapeutic strategy in human tumors expressing high levels of Δ40p53.