The Neurod1/4-Ntrk3-Src pathway regulates gonadotrope cell adhesion and motility.

Pituitary gonadotrope cells are essential for the endocrine regulation of reproduction in vertebrates. These cells emerge early during embryogenesis, colonize the pituitary glands and organize in tridimensional networks, which are believed to be crucial to ensure proper regulation of fertility. However, the molecular mechanisms regulating the organization of gonadotrope cell population during embryogenesis remain poorly understood. In this work, we characterized the target genes of NEUROD1 and NEUROD4 transcription factors in the immature gonadotrope αT3-1 cell model by in silico functional genomic analyses. We demonstrated that NEUROD1/4 regulate genes belonging to the focal adhesion pathway. Using CRISPR/Cas9 knock-out approaches, we established a double NEUROD1/4 knock-out αT3-1 cell model and demonstrated that NEUROD1/4 regulate cell adhesion and cell motility. We then characterized, by immuno-fluorescence, focal adhesion number and signaling in the context of NEUROD1/4 insufficiency. We demonstrated that NEUROD1/4 knock-out leads to an increase in the number of focal adhesions associated with signaling abnormalities implicating the c-Src kinase. We further showed that the neurotrophin tyrosine kinase receptor 3 NTRK3, a target of NEUROD1/4, interacts physically with c-Src. Furthermore, using motility rescue experiments and time-lapse video microscopy, we demonstrated that NTRK3 is a major regulator of gonadotrope cell motility. Finally, using a Ntrk3 knock-out mouse model, we showed that NTRK3 regulates gonadotrope cells positioning in the developing pituitary, in vivo. Altogether our study demonstrates that the Neurod1/4-Ntrk3-cSrc pathway is a major actor of gonadotrope cell mobility, and thus provides new insights in the regulation of gonadotrope cell organization within the pituitary gland.

SlitC-PlexinA1 mediates iterative inhibition for orderly passage of spinal commissural axons through the floor plate.

Spinal commissural axon navigation across the midline in the floor plate requires repulsive forces from local Slit repellents. The long-held view is that Slits push growth cones forward and prevent them from turning back once they became sensitized to these cues after midline crossing. We analyzed with fluorescent reporters Slits distribution and FP glia morphology. We observed clusters of Slit-N and Slit-C fragments decorating a complex architecture of glial basal process ramifications. We found that PC2 proprotein convertase activity contributes to this pattern of ligands. Next, we studied Slit-C acting via PlexinA1 receptor shared with another FP repellent, the Semaphorin3B, through generation of a mouse model baring PlexinA1Y1815F mutation abrogating SlitC but not Sema3B responsiveness, manipulations in the chicken embryo, and ex vivo live imaging. This revealed a guidance mechanism by which SlitC constantly limits growth cone exploration, imposing ordered and forward-directed progression through aligned corridors formed by FP basal ramifications.

A Spatiotemporal Sequence of Sensitization to Slits and Semaphorins Orchestrates Commissural Axon Navigation.

Accurate perception of guidance cues is crucial for cell and axon migration. During initial navigation in the spinal cord, commissural axons are kept insensitive to midline repellents. Upon midline crossing in the floor plate, they switch on responsiveness to Slit and Semaphorin repulsive signals and are thus propelled away and prevented from crossing back. Whether and how the different midline repellents control specific aspects of this navigation remain to be elucidated. We set up a paradigm for live-imaging and super-resolution analysis of PlexinA1, Neuropilin-2, and Robo1/2 receptor dynamics during commissural growth cone navigation in chick and mouse embryos. We uncovered a remarkable program of sensitization to midline cues achieved by unique spatiotemporal sequences of receptor allocation at the growth-cone surface that orchestrates receptor-specific growth-cone behavior changes. This reveals post-translational mechanisms whereby coincident guidance signals are temporally resolved to allow the generation of specific guidance responses.

Commissural axon navigation in the spinal cord: A repertoire of repulsive forces is in command.

The navigation of commissural axons in the developing spinal cord has attracted multiple studies over the years. Many important concepts emerged from these studies which have enlighten the general mechanisms of axon guidance. The navigation of commissural axons is regulated by a series of cellular territories which provides the diverse guidance information necessary to ensure the successive steps of their pathfinding towards, across, and away from the ventral midline. In this review, we discuss how repulsive forces, by propelling, channelling, and confining commissural axon navigation, bring key contributions to the formation of this neuronal projection.

The Proto-oncogene c-Kit Inhibits Tumor Growth by Behaving as a Dependence Receptor.

c-Kit is a classic proto-oncogene either mutated or upregulated in cancer cells, and this leads to its constitutive kinase activation and, thus, to uncontrolled proliferation. Although the pro-oncogenic role of c-Kit is of no doubt, some observations do not fit well with c-Kit solely as a tumor-promoting moiety. We show here that c-Kit actively triggers cell death in various cancer cell lines unless engaged by its ligand stem cell factor (SCF). This pro-death activity is enhanced when the kinase activation of c-Kit is silenced and is due to c-Kit intracellular cleavage by caspase-like protease at D816. Moreover, in vivo, overexpression of a c-Kit kinase-dead mutant inhibits tumor growth, and this intrinsic c-Kit tumor-suppressive activity is dependent on the D816 cleavage. Thus, c-Kit acts both as a proto-oncogene via its kinase activity and as a tumor suppressor via its dependence receptor activity.

The dependence receptor TrkC regulates the number of sensory neurons during DRG development.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.

Hey1- and p53-dependent TrkC proapoptotic activity controls neuroblastoma growth.

The neurotrophin-3 (NT-3) receptor tropomyosin receptor kinase C (TrkC/NTRK3) has been described as a dependence receptor and, as such, triggers apoptosis in the absence of its ligand NT-3. This proapoptotic activity has been proposed to confer a tumor suppressor activity to this classic tyrosine kinase receptor (RTK). By investigating interacting partners that might facilitate TrkC-induced cell death, we have identified the basic helix-loop-helix (bHLH) transcription factor Hey1 and importin-α3 (karyopherin alpha 4 [KPNA4]) as direct interactors of TrkC intracellular domain, and we show that Hey1 is required for TrkC-induced apoptosis. We propose here that the cleaved proapoptotic portion of TrkC intracellular domain (called TrkC killer-fragment [TrkC-KF]) is translocated to the nucleus by importins and interacts there with Hey1. We also demonstrate that Hey1 and TrkC-KF transcriptionally silence mouse double minute 2 homolog (MDM2), thus contributing to p53 stabilization. p53 transcriptionally regulates the expression of TrkC-KF cytoplasmic and mitochondrial interactors cofactor of breast cancer 1 (COBRA1) and B cell lymphoma 2-associated X (BAX), which will subsequently trigger the intrinsic pathway of apoptosis. Of interest, TrkC was proposed to constrain tumor progression in neuroblastoma (NB), and we demonstrate in an avian model that TrkC tumor suppressor activity requires Hey1 and p53.

Microenvironment-Driven Shift of Cohesion/Detachment Balance within Tumors Induces a Switch toward Metastasis in Neuroblastoma.

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. Disseminated forms have high frequency of multiple tumoral foci whose etiology remains unknown; NB embryonic origin limits investigations in patients and current models. We developed an avian embryonic model driving human NB tumorigenesis in tissues homologous to patients. We found that aggressive NBs display a metastatic mode, secondary dissemination via peripheral nerves and aorta. Through tumor transcriptional profiling, we found that NB dissemination is induced by the shutdown of a pro-cohesion autocrine signal, SEMA3C, which constrains the tumoral mass. Lowering SEMA3C levels shifts the balance toward detachment, triggering NB cells to collectively evade the tumor. Together with patient cohort analysis, this identifies a microenvironment-driven pro-metastatic switch for NB.

Dependence receptors and colorectal cancer.

The research on colorectal cancer (CRC) biology has been leading the oncology field since the early 1990s. The search for genetic alterations has allowed the identification of the main tumour suppressors or oncogenes. Recent work obtained in CRC has unexpectedly proposed the existence of novel category of tumour suppressors, the so-called 'dependence receptors'. These transmembrane receptors behave as Dr Jekyll and Mr Hyde with two opposite sides: they induce a positive signalling (survival, proliferation, differentiation) in presence of their ligand, but are not inactive in the absence of their ligand and rather trigger apoptosis when unbound. This trait confers them a conditional tumour suppressor activity: they eliminate cells that grow abnormally in an environment offering a limited quantity of ligand. This review will describe how receptors such as deleted in colorectal carcinoma (DCC), uncoordinated 5 (UNC5), rearranged during transfection (RET) or TrkC constrain CRC progression and how this dependence receptor paradigm may open up therapeutical perspectives.

The dependence receptor TrkC triggers mitochondria-dependent apoptosis upon Cobra-1 recruitment.

The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. The molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic "killer" fragment (TrkC KF). Here, we show that TrkC KF interacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. We also show that, in the developing chick neural tube, NT-3 silencing is associated with neuroepithelial cell death that is rescued by Cobra1 silencing. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes Bax activation, cytochrome c release, and apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1.

Dependence receptor TrkC is a putative colon cancer tumor suppressor.

The TrkC neurotrophin receptor belongs to the functional dependence receptor family, members of which share the ability to induce apoptosis in the absence of their ligands. Such a trait has been hypothesized to confer tumor-suppressor activity. Indeed, cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC is a classic tyrosine kinase receptor and therefore generally considered to be a proto-oncogene. We show here that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Finally, we provide evidence that a mutation of TrkC detected in a sporadic cancer is a loss-of-proapoptotic function mutation. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor.

Neurotrophins and cell death.

The neurotrophins - NGF, BDNF, NT-3 - are secreted proteins that play a major role in neuron survival, differentiation and axon wiring toward target territories. They do so by interacting with their main tyrosine kinase receptors TrkA, TrkB, TrkC and p75(NTR). Even though there is a general consensus on the view that neurotrophins are survival factors, there are two fundamentally different views on how they achieve this survival activity. One prevailing view is that all neurons and more generally all normal cells are naturally committed to die unless a survival factor blocks this death. This death results from the engagement of a "default" apoptotic cell program. The minority report supports, on the opposite, that neurotrophin withdrawal is associated with an active signal of cell death induced by unbound dependence receptors. We will discuss here how neurotrophins regulate cell death and survival and how this has implications not only during nervous system development but also during cancer progression.

Targeting neurotrophin-3 and its dependence receptor tyrosine kinase receptor C: a new antitumoral strategy.

INTRODUCTION: Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor that belongs to the tyrosine kinase receptor family. This family primarily consists of proto-oncogenes, and TrkC has been involved in oncogenic translocations. However, its expression in tumors is often associated with good prognosis, suggesting it actually acts as a tumor suppressor. TrkC has recently been demonstrated to be a dependence receptor, which regulates neuronal survival. Dependence receptors share the ability to trigger apoptosis in the absence of their ligand, a feature that has been suggested to confer a tumor suppressor function to these receptors. A selective advantage for a tumor cell to survive in an environment with limited ligand availability would hence be either to lose the expression of the dependence receptor, or to gain expression of its ligand. AREAS COVERED: The role of neurotrophin-3 (NT-3) and its dependence receptor TrkC in neuroblastoma, and its suitability as a therapeutic target. EXPERT OPINION: Autocrine production of NT-3 represents a selective advantage for tumor growth and dissemination, in a large fraction of aggressive neuroblastoma. Disruption of the NT-3 autocrine loop in malignant neuroblasts, triggers neuroblastoma cell death, and inhibits neuroblastoma metastasis in animal models. Thus, a novel way of targeting the tyrosine kinase receptor, is via the reactivation of its intrinsic ability to trigger cell death.

RET modulates cell adhesion via its cleavage by caspase in sympathetic neurons.

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.

Neurotrophin-3 production promotes human neuroblastoma cell survival by inhibiting TrkC-induced apoptosis.

Tropomyosin-related kinase receptor C (TrkC) is a neurotrophin receptor with tyrosine kinase activity that was expected to be oncogenic. However, it has several characteristics of a tumor suppressor: its expression in tumors has often been associated with good prognosis; and it was recently demonstrated to be a dependence receptor, transducing different positive signals in the presence of ligand but inducing apoptosis in the absence of ligand. Here we show that the TrkC ligand neurotrophin-3 (NT-3) is upregulated in a large fraction of aggressive human neuroblastomas (NBs) and that it blocks TrkC-induced apoptosis of human NB cell lines, consistent with the idea that TrkC is a dependence receptor. Functionally, both siRNA knockdown of NT-3 expression and incubation with a TrkC-specific blocking antibody triggered apoptosis in human NB cell lines. Importantly, disruption of the NT-3 autocrine loop in malignant human neuroblasts triggered in vitro NB cell death and inhibited tumor growth and metastasis in both a chick and a mouse xenograft model. Thus, we believe that our data suggest that NT-3/TrkC disruption is a putative alternative targeted therapeutic strategy for the treatment of NB.

The TrkC receptor induces apoptosis when the dependence receptor notion meets the neurotrophin paradigm.

The TrkC/NT-3 receptor/ligand pair is believed to be part of the classic neurotrophic theory claiming that neuronal death occurs by default when neurotrophic factors become limited, through loss of survival signals. Here, we show that TrkC is a dependence receptor and, as such, induces caspase-dependent apoptotic death in the absence of NT-3 in immortalized cells, a proapoptotic activity inhibited by the presence of NT-3. This proapoptotic activity of TrkC relies on the caspase-mediated cleavage of the intracellular domain of TrkC, which permits the release of a proapoptotic fragment. This fragment induces apoptosis through a caspase-9-dependent mechanism. Finally, we show that the death of dorsal root ganglion (DRG) neurons provoked by NT-3 withdrawal is inhibited when TrkC-proapoptotic activity is antagonized. Thus, the death of neurons upon disappearance of NT-3 is not only due to a loss of survival signals but also to the active proapoptotic activity of the unbound TrkC dependence receptor.

RGM and its receptor neogenin regulate neuronal survival.

Repulsive guidance molecule (RGM) is an axon guidance protein that repels retinal axons upon activation of the neogenin receptor. To understand the functions of RGM-neogenin complexes in vivo, we used gene transfer technology to perturb their expression in the developing neural tube of chick embryos. Surprisingly, neogenin over-expression or RGM down-expression in the neural tube induces apoptosis. Neogenin pro-apoptotic activity in immortalized neuronal cells and in the neural tube is associated with the cleavage of its cytoplasmic domain by caspases. Thus neogenin is a dependence receptor inducing cell death in the absence of RGM, whereas the presence of RGM inhibits this effect.

Binding of the Drosophila cytokine Spätzle to Toll is direct and establishes signaling.

The extracellular protein Spätzle is required for activation of the Toll signaling pathway in the embryonic development and innate immune defense of Drosophila. Spätzle is synthesized as a pro-protein and is processed to a functional form by a serine protease. We show here that the mature form of Spätzle triggers a Toll-dependent immune response after injection into the hemolymph of flies. Spätzle specifically bound to Drosophila cells and to Cos-7 cells expressing Toll. Furthermore, in vitro experiments showed that the mature form of Spätzle bound to the Toll ectodomain with high affinity and with a stoichiometry of one Spätzle dimer to two receptors. The Spätzle pro-protein was inactive in all these assays, indicating that the pro-domain sequence, which is natively unstructured, acts to prevent interaction of the cytokine and its receptor Toll. These results show that, in contrast to the human Toll-like receptors, Drosophila Toll requires only an endogenous protein ligand for activation and signaling.

Drosophila MyD88 is required for the response to fungal and Gram-positive bacterial infections.

We report here the identification and functional characterization of DmMyD88, a gene encoding the Drosophila homolog of mammalian MyD88. DmMyD88 combines a Toll-IL-1R homology (TIR) domain and a death domain. Overexpression of DmMyD88 was sufficient to induce expression of the antifungal peptide Drosomycin, and induction of Drosomycin was markedly reduced in DmMyD88-mutant flies. DmMyD88 interacted with Toll through its TIR domain and required the death domain proteins Tube and Pelle to activate expression of Drs, which encodes Drosomycin. DmMyD88-mutant flies were highly susceptible to infection by fungi and Gram-positive bacteria, but resisted Gram-negative bacterial infection much as did wild-type flies. Phenotypic comparison of DmMyD88-mutant flies and MyD88-deficient mice showed essential differences in the control of Gram-negative infection in insects and mammals.