Growing axons are guided to their targets by attractive and repulsive cues. In the developing spinal cord, Netrin-1 and Shh guide commissural axons toward the midline. However, the combined ...inhibition of their activity in commissural axon turning assays does not completely abrogate turning toward floor plate tissue, suggesting that additional guidance cues are present. Here we show that the prototypic angiogenic factor VEGF is secreted by the floor plate and is a chemoattractant for commissural axons in vitro and in vivo. Inactivation of Vegf in the floor plate or of its receptor Flk1 in commissural neurons causes axon guidance defects, whereas Flk1 blockade inhibits turning of axons to VEGF in vitro. Similar to Shh and Netrin-1, VEGF-mediated commissural axon guidance requires the activity of Src family kinases. Our results identify VEGF and Flk1 as a novel ligand/receptor pair controlling commissural axon guidance.
► VEGF is secreted by the floor plate ► Haplodeficiency of Vegf in the floor plate causes axon guidance defects in vivo ► Inactivation of Flk1 in commissural neurons causes axon guidance defects in vivo ► VEGF/Flk1 activates Src family kinases and induces commissural axon turning in vitro
In the hematopoietic system, Syk family tyrosine kinases are essential components of immunoreceptor ITAM-based signaling. While there is increasing data indicating the involvement of immunoreceptors ...in neural functions, the contribution of Syk kinases remains obscure. Previously, we identified phosphorylated forms of Syk kinases in specialized populations of migrating neurons or projecting axons. Moreover, we identified ephrin/Eph as guidance molecules utilizing the ITAM-bearing CD3zeta (Cd247) and associated Syk kinases for the growth cone collapse response induced in vitro Here, we show that in the developing spinal cord, Syk is phosphorylated in navigating commissural axons. By analyzing axon trajectories in open-book preparations of Syk(-/-); Zap70(-/-) mouse embryos, we show that Syk kinases are dispensable for attraction towards the midline but confer growth cone responsiveness to repulsive signals that expel commissural axons from the midline. Known to serve a repulsive function at the midline, ephrin B3/EphB2 are obvious candidates for driving the Syk-dependent repulsive response. Indeed, Syk kinases were found to be required for ephrin B3-induced growth cone collapse in cultured commissural neurons. In fragments of commissural neuron-enriched tissues, Syk is in a constitutively phosphorylated state and ephrin B3 decreased its level of phosphorylation. Direct pharmacological inhibition of Syk kinase activity was sufficient to induce growth cone collapse. In conclusion, Syk kinases act as a molecular switch of growth cone adhesive and repulsive responses.
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 PlexinA1
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.
Abstract Over the last decade, the success of immunotherapy in treating numerous solid and hematological tumors is unprecedent. However, the poor preclinical predictive power of current model systems ...explains in part the failures during late-stages clinical drug development for the numerous potential immunotherapies. Thus, there is a crucial need for innovative technologies offering fast and reliable evaluation of immunotherapies on patient-derived models. Using its proprietary process of micro-implantation of patient tumor cells within selected organs of series of avian embryos, Oncofactory developed two humanized avian xenograft models, namely IMMUNO-AVI-patient derived xenograft (iAVI-PDXTM) and IMMUNO-AVI-cell line derived xenograft (iAVI-cellDXTM). These models, consisting in the co-implantation of human peripheral-blood mononuclear cells (PBMC) and human cancer cells were validated through a sequence of experimental steps. First, we found that transplantation of PBMCs into avian tissues exhibited good survival and low allogenicity against the avian embryonic organism. Second, in embryos engrafted with tumoral cells, we established that intravenously injected PBMCs successfully populated the tumor. Third, to reduce the dilution of immune cells in the embryonic tissues and to better standardize the number of implanted cells in between embryos, we optimized the process by direct co-implantation of both PBMCs and cancer cells. We analyzed the immune cell contingent sorted from dissected tumors and found that all major immune cells populations, including T and B lymphocytes, monocytes as well as NK cells were preserved post-grafting, when compared to pre-grafted PBMCs. The AVI-cellDXTM and AVI-PDXTM models initially developed by Oncofactory allows fast and reproducible tumor growth with statistical evaluation of molecule efficacy already at 24 hours post-injection. To assess whether our humanized models retain these advantages, we analyzed the outcome of anti-PD1 antibody (Pembrolizumab) intravenous injection in series of avian embryos co-engrafted with human PBMCs and PDL1-expressing breast and colorectal cancer cell lines. Twenty-four hours post vehicle or Pembrolizumab exposure, we analyzed the tumor volumes in grafted embryos using light sheet microscopy. In all three cancer models, we found a statistically significant reduction of the volume upon pembrolizumab, compared with vehicle. Using a panel of molecular markers in flow cytometry, we found that the Pembrolizumab efficiently targeted T lymphocytes in the tumors. Finally, we validated the efficiency of Pembrolizumab treatment in models of co-implantation of human PBMCs with melanoma and colorectal patient samples. Thus, humanized iAVI-cellDXTM or -PDXTM technology bring powerful and fast alternative to mouse models, to strongly accelerate preclinical developments in immuno-oncology. Citation Format: Marjorie Lacourrege, Clélia Costechareyre, Loraine Jarrosson, Romain Teinturier, Frédéric Berget, Céline Delloye-Bourgeois, Valérie Castellani. The immuno-AVI-PDX, am innovative patient-derived xenograft model for preclinical immuno-oncology studies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1459.
During development, axons migrate long distances in responses to attractive or repulsive signals that are detected by their growth cones. One of these signals is mediated by netrin-1, a diffusible ...laminin-related molecule that both attracts and repels growth cones via interaction with its receptor DCC (deleted in colorectal cancer). Here we show that DCC in both commissural neurons and immortalized cells, is partially associated with cholesterol- and sphingolipid-enriched membrane domains named lipid rafts. This localization of DCC in lipid rafts is mediated by the palmitoylation within its transmembrane region. Moreover, this raft localization of DCC is required for netrin-1-induced DCC-dependent ERK activation, and netrin-1-mediated axon outgrowth requires lipid raft integrity. Thus, the presence of axon guidance-related receptors in lipid rafts appears to be a crucial pre-requisite for growth cone response to chemo-attractive or repulsive cues.
Commissural circuits are brain and spinal cord connections which interconnect the two sides of the central nervous system (CNS). They play essential roles in brain and spinal cord processing, ...ensuring left-right coordination and synchronization of information and commands. During the formation of neuronal circuits, all commissural neurons of the central nervous system must accomplish a common task, which is to project their axon onto the other side of the nervous system, across the midline that delineates the two halves of the CNS. How this task is accomplished has been the topic of extensive studies over the last past 20 years and remains one of the best models to investigate axon guidance mechanisms. In the first part of this review, I will introduce the commissural circuits, their general role in the physiology of the nervous system, and their recognized or suspected pathogenic properties in human diseases. In the second part of the review, I will concentrate on two commissural circuits, the spinal commissures and the corpus callosum, to detail the cellular and molecular mechanisms governing their formation, mostly during their navigation at the midline.
Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the ...dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2
while caveolin-1
nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75
, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75
that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75
) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.
Control of semaphorin signaling Castellani, Valérie; Rougon, Geneviève
Current Opinion in Neurobiology,
10/2002, Letnik:
12, Številka:
5
Book Review, Journal Article
Recenzirano
Receptor complexes for the chemorepellent factors of the semaphorin family activate intracellular pathways that trigger actin rearrangements underlying growth cone collapse and repellent behavior. ...Some evidence has been provided for a complex and dynamic pattern of interaction between members of the small Rho guanosine triphosphatases and plexin proteins that are the receptor subunits responsible for initiating semaphorin signaling. The characterization of new components of semaphorin receptor complexes, the implication of several distinct classes of cytoplasmic effectors, together with the observation of a variety of processes modulating the semaphorin signal have provided a basis for a much improved, but still intricate view of the semaphorin transduction pathways in neurons.
Recent experiments have provided insights into the molecular machinery that transduces semaphorin signals and into the mechanisms modulating cellular responses to semaphorins in the nervous system.
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