Neurofilament heavy chain (NEFH) gene was recently identified to cause autosomal dominant axonal Charcot-Marie-Tooth disease (CMT2cc). However, the clinical spectrum of this condition and the ...physio-pathological pathway remain to be delineated. We report 12 patients from two French families with axonal dominantly inherited form of CMT caused by two new mutations in the NEFH gene. A remarkable feature was the early involvement of proximal muscles of the lower limbs associated with pyramidal signs in some patients. Nerve conduction velocity studies indicated a predominantly motor axonal neuropathy. Unique deletions of two nucleotides causing frameshifts near the end of the NEFH coding sequence were identified: in family 1, c.3008_3009del (p.Lys1003Argfs*59), and in family 2 c.3043_3044del (p.Lys1015Glyfs*47). Both frameshifts lead to 40 additional amino acids translation encoding a cryptic amyloidogenic element. Consistently, we show that these mutations cause protein aggregation which are recognised by the autophagic pathway in motoneurons and triggered caspase 3 activation leading to apoptosis in neuroblastoma cells. Using electroporation of chick embryo spinal cord, we confirm that NEFH mutants form aggregates in vivo and trigger apoptosis of spinal cord neurons. Thus, our results provide a physiological explanation for the overlap between CMT and amyotrophic lateral sclerosis (ALS) clinical features in affected patients.
In humans, defects of the corticospinal tract have been attributed to mutations in the gene encoding L1 CAM, a phenotype that is reproduced in L1-deficient mice. Using coculture assays, we report ...that Sema3A secreted from the ventral spinal cord repels cortical axons from wild-type but not from L1-deficient mice. L1 and neuropilin-1 (NP-1) form a stable complex, and their extracellular domains can directly associate. Thus, L1 is a component of the Sema3A receptor complex, and L1 mutations may disrupt Sema3A signaling in the growth cone, leading to guidance errors. Addition of soluble L1Fc chimeric molecules does not restore Sema3A responsiveness of L1-deficient axons; instead, it converts the repulsion of wild-type axons into an attraction, further supporting a function for L1 in the Sema3A transducing pathways within the growth cone.
During axon navigation, Semaphorin3A-induced growth cone retraction is correlated with endocytosis. Although its function remains elusive, we showed previously that the cell adhesion molecule of the ...immunoglobulin super family L1 associates with Neuropilin-1 (NP-1) the Sema3A-binding subunit of the receptor complex and is required for Sema3A to elicit axonal repulsive responses. We report here that upon Sema3A binding to NP-1, L1 and NP-1 are co-internalized through a clathrin-dependent mechanism mediated by L1. We show that in COS7 cells, L1/NP-1 endocytosis is correlated with a cell contraction similar to that observed with the Plexin (Plex)/NP-1 or Plex/NP1/L1 complexes. In neuronal cultures, a L1-mimetic peptide able to switch Sema3A repulsive responses to attraction blocks both endocytosis and growth cone collapse. Similarly, in the COS7 cell model, peptide application prevents both the Sema3-induced L1/NP-1 internalization and cell collapse. These studies demonstrate that the L1/NP-1 complex is able to confer a biological response to Sema3A with L1 mediating receptor internalization following ligand activation. They also reveal that endocytosis controlled by L1/NP-1
cis and
trans interactions is pivotal in Sema3A-mediated axon guidance.
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.
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•We set an animal model fully replicating human NB tumorigenesis and dissemination•Stage 4 NB foci arise from metastatic dissemination via peripheral nerves and aorta•RNA-seq data reveal an NB gene program regulated by the embryonic microenvironment•Sema3C signaling shutdown in NB primary tumors is a master pro-metastatic trigger
Delloye-Bourgeois et al. developed an aggressive human neuroblastoma (NB) model in which NB cells disseminate via peripheral nerves and aorta by placing the cells at their primitive environment in avian embryos. NB dissemination is regulated by a cohesion signaling controlled by SEMA3C via NRP/PLXN complexes.
During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ...ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons(1). These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues(2,3). Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FP(cm))(4-6). We then exemplify the use of this FP(cm) in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FP(cm). Third, the tissue are processed for Western blot analysis of commissural markers.
During development, axon guidance receptors play a crucial role in regulating axons sensitivity to both attractive and repulsive cues. Indeed, activation of the guidance receptors is the first step ...of the signaling mechanisms allowing axon tips, the growth cones, to respond to the ligands. As such, the modulation of their availability at the cell surface is one of the mechanisms that participate in setting the growth cone sensitivity. We describe here a method to precisely visualize the spatio-temporal cell surface dynamics of an axon guidance receptor both in vitro and in vivo in the developing chick spinal cord. We took advantage of the pH-dependent fluorescence property of a green fluorescent protein (GFP) variant to specifically detect the fraction of the axon guidance receptor that is addressed to the plasma membrane. We first describe the in vitro validation of such pH-dependent constructs and we further detail their use in vivo, in the chick spinal chord, to assess the spatio-temporal dynamics of the axon guidance receptor of interest.
Previously, we demonstrated that loss of SEMA3F, a secreted semaphorin encoded in 3p21.3, is associated with higher stages in lung cancer and primary tumor cells studied with anti-vascular ...endothelial growth factor (VEGF) and SEMA3F antibodies. In vitro, SEMA3F inhibits cell spreading; this activity is opposed by VEGF. These results suggest that VEGF and SEMA3F compete for binding to their common neuropilin receptor. In the present report, we investigated the attractive/repulsive effects of SEMA3F on cell migration when cells were grown in a threedimensional system and exposed to a SEMA3F gradient. In addition, we adapted the neurobiologic stripe assay to analyze the migration of tumor cells in response to SEMA3F. In the motile breast cancer cell line C100, which expresses both neuropilin-1 (NRPi) and neuropilin-2 (NRP2) receptors, SEMA3F had a repulsive effect, which was blocked by anti-NRP2 antibody. In less motile MCF7 cells, which express only NRPi, SEMA3F inhibited cell contacts with loss of membrane-associated E-cadherin and β-catenin without motility induction. Cell spreading and proliferation were reduced. These results support the concept that in a first step during tumorigenesis, normal tissues expressing SEMA3F would try to prevent tumor cells from spreading and attaching to the stroma for further implantation.
The Neurotrophic factor gdnf plays diverse developmental roles, supporting survival and also acting as a chemoattractant for axon and cell migration. We report that in the developing spinal cord, a ...focal source of gdnf is present in the floor plate (FP) where commissural axons cross the midline. Gdnf has no direct guidance properties but switches on the responsiveness of crossing commissural growth cones to the midline repellent Semaphorin3B by suppressing calpain-mediated processing of the Sema3B signaling coreceptor Plexin-A1. Analysis of single and double mutant mouse models indicates that although gdnf is the principal trigger of Sema3B midline repulsion, it acts with another FP cue, NrCAM. Finally, genetic and in vitro experiments provide evidence that this gdnf effect is RET independent and mediated by NCAM/GFRα1 signaling. This study identifies a regulator of midline crossing and reveals interplays between Semaphorin and gdnf signaling during axon guidance.
► gdnf is expressed in the floor plate of the developing spinal cord ► gdnf deficiency in mouse embryos induces defective commissural axon guidance ► gdnf switches on axon responsiveness to the midline repellent Semaphorin3B ► gdnf acts through NCAM/GFRα1 and upregulates Plexin-A1 by suppressing calpain activity
Commissural axon guidance requires repulsive midline signaling activated at postcrossing stage to control floor plate exit. Here, Charoy et al. show that a floor plate gdnf source switches on commissural responsiveness to Semaphorin3B, acting through NCAM-GFRα1 to set Plexin-A1 at the growth cone surface.
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.
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•Live monitoring of guidance receptors reveals specific dynamics in commissural axons•Slit and Semaphorin receptors have different temporal patterns of membrane insertion•A front-rear polarity organizes PlxnA1 and Robo1 receptors at the growth-cone surface•Robo1, but not Robo2, is sorted during midline crossing
Pignata et al. developed a setup to monitor the dynamics of receptors for midline repellents during commissural axon navigation across the floor plate. They find specific spatiotemporal patterns of receptor insertion at the growth-cone membrane. This suggests that differences of receptor dynamics can elicit functional specificities of midline repellents.
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