Glioblastoma multiforme (GBM) are highly invasive and angiogenic malignancies with a median survival time from diagnosis of <15 months. Previous work has revealed robust overexpression of fibronectin ...(FN) mRNA in GBM, although immunohistochemical staining of FN in these tumors is typically associated with the angiogenic vasculature. Here we sought to examine the expression of tumor cell FN and address its possible involvement in the invasive phenotype of GBM. We found that FN was expressed and assembled into fibrillar arrays in human tumors and in established GBM lines. Cultured cells spontaneously formed dense cellular networks and spheroid-like domes. Depletion of FN by targeted-short hairpin RNA expression disrupted matrix assembly and multicellular network organization by exerting profound effects on cell adhesion and motility. Although FN depletion enhanced persistent directional migration of single cells, it compromised collective invasion of spheroids through a laminin-rich matrix and sensitized cells to ionizing radiation. In orthotopic grafts, FN depletion significantly reduced tumor growth and angiogenesis. Together our results show that FN produced by the tumor cells has a role in GBM pathophysiology and they provide insights into the implications that targeting FN interactions may have for combating this dreaded disease.
Immunoglobulin superfamily (IgSF) proteins are implicated in diverse steps of brain development, including neuronal migration, axon pathfinding, target recognition and synapse formation, as well as ...in the maintenance and function of neuronal networks in the adult. We provide here a review of recent findings on the diversity and the role of transmembrane and secreted members of IgSF proteins in the nervous system. We illustrate that the complexity of IgSF protein function results from various different levels of regulation including regulation of gene expression, protein localization, and protein interactions.
Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this ...activity to axon pathfinding is still unclear. Using genetic and culture models, we provide evidence that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, we show that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue.
Mutations in the L1 gene induce a spectrum of human neurological disorders due to abnormal development of several brain structures and fiber tracts. Among its binding partners, L1 immunoglobulin ...superfamily adhesion molecule (Ig CAM) associates with neuropilin‐1 (NP‐1) to form a semaphorin3A (Sema3A) receptor and soluble L1 converts Sema3A‐induced axonal repulsion into attraction. Using L1 constructs containing missense pathological mutations, we show here that this reversion is initiated by a specific trans binding of L1 to NP‐1, but not to L1 or other Ig CAMs, and leads to activation of the NO/cGMP pathway. We identified the L1–NP‐1‐binding site in a restricted sequence of L1 Ig domain 1, as a peptide derived from this region could reverse Sema3A repulsive effects. A pathological L1 missense mutation located in this sequence specifically disrupts both L1–NP‐1 complex formation and Sema3A reversion, suggesting that the cross‐talk between L1 and Sema3A might participate in human brain development.
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.
The neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) contribute to long-term potentiation (LTP) in the CA1 hippocampus. Here we report that the deficient LTP found in ...slices prepared from NCAM knockout mice and in organotypic slice cultures treated with Endo-N, an enzyme that cleaves the PSA moiety of NCAM, can be rescued by brain-derived neurotrophic factor (BDNF). This effect is not reproduced by nerve growth factor, but can be obtained with high concentrations of NT4/5. The effect of BDNF cannot be accounted for by modifications of N-methyl-D-aspartate receptor-dependent responses or of high-frequency bursts. PSA-NCAM, however, could directly interact with BDNF. Exogenous application of PSA residues or recombinant PSA-NCAM also prevents LTP. Furthermore trkB phosphorylation, and thus BDNF signaling, is reduced in both NCAM knockout mice and Endo-N-treated slice cultures. These results suggest that one action of PSA-NCAM could be to sensitize pyramidal neurons to BDNF, thereby modulating activity-dependent synaptic plasticity.
Many factors have been shown to promote myelination, but few have been shown to be inhibitory. Here, we show that polysialylated-neural cell adhesion molecule (PSA-NCAM) can negatively regulate ...myelin formation. During development, PSA-NCAM is first expressed on all growing fibers; then, axonal expression is down-regulated and myelin deposition occurs only on PSA-NCAM-negative axons. Similarly, in cocultures of oligodendrocytes and neurons, PSA-NCAM expression on axons is initially high, but decreases as myelination proceeds. Importantly, if expression of PSA-NCAM is prematurely decreased in cultures, by either antibody-mediated internalization or enzymatic removal of the PSA moieties with endoneuraminidase N (endo-N), myelination increases 4- to 5-fold. In the optic nerve, premature cleavage of PSA moieties by intravitreous injection of endo-N also induces a transient increase in the number of myelinated internodes, but does not interfere with the onset of myelination. Previously, we showed that axonal electrical activity strongly induced myelination, which could be prevented by tetrodotoxin (TTX), an action potential blocker. Interestingly, removal of PSA moieties does not reverse the inhibition of myelination by TTX. Together, this suggests that myelination is tightly controlled by both positive (electrical activity) and negative (PSA-NCAM expression) regulatory signals.
Objectives: The involvement of skeletal muscle mitochondrial uncoupling protein-3 (UCP3) in the control of energy expenditure in skeletal muscle and at the whole-body level is still a matter of ...debate. We previously reported that UCP3 downregulation is linked to an enhanced mitochondrial energy metabolism in rat skeletal muscle as a result of acute capsiate treatment. Here, we aimed at investigating noninvasively the effects of chronic capsiate ingestion on metabolic changes occurring in exercising gastrocnemius muscle and at the whole-body level. Methods: We used an original experimental setup allowing a complete noninvasive investigation of gastrocnemius muscle function in situ using 31-phosphorus magnetic resonance spectroscopy. Whole-body fat composition was determined using magnetic resonance imaging and UCP3 gene expression was measured by quantitative real-time RT-PCR analysis. Results: We found that a 14-day daily administration of capsiate (100 mg kg-1 body weight) reduced UCP3 gene expression and increased phosphocreatine level at baseline and during the stimulation period in gastrocnemius muscle. During muscle stimulation, pHi showed a larger alkalosis in the capsiate group suggesting a lower glycolysis and a compensatory higher aerobic contribution to ATP production. Although the capsiate-treated rats were hyperphagic as compared to control animals, they showed a lower weight gain coupled to a decreased abdominal fat content. Conclusion: Overall, our data indicated that capsiate administration contributes to the enhancement of aerobic ATP production and the reduction of body fat content coupled to a UCP3 gene downregulation.
We show that the loss or inactivation of the polysialic acid (PSA) tail of neural cell adhesion molecule (NCAM) on rat cortical neurons in culture leads to reduced differentiation and survival. The ...mechanism by which this negative effect is mediated appears to involve the neuronal response to brain‐derived neurotrophic factor (BDNF): (i) in the absence of PSA or in the presence of excess free PSA added to the culture medium, BDNF‐induced cell signalling is reduced; (ii) the addition of exogenous BDNF to the medium reverses the effect of PSA loss or inactivation. These data suggest that PSA‐NCAM, previously shown to modulate cell migration and plasticity, is needed for an adequate sensitivity of neurons to BDNF.
mCD24, a glycosylphosphatidylinositol-anchored highly glycosylated molecule, is expressed on differentiating neurons during development. In the adult CNS, its expression is restricted to immature ...neurons located in two regions showing ongoing neurogenesis: the subventricular zone (SVZ) of the lateral ventricle pathway and the dentate gyrus (DG) of the hippocampal formation. Here, combining bromodeoxyuridine (BrdU) and proliferating cell nuclear antigen labelings we confirmed that mCD24 is expressed on proliferating cells. To determine whether the inactivation of the molecule may affect adult neurogenesis, we analyzed the phenotype of mCD24-deficient mice (mCD24-/-). We labeled cells in S-phase with a pulse, a long, or a cumulative administration of BrdU and analyzed cells in different zones according to their dividing rate (rapid and slow) both in the control and mCD24-/-. We found a significant increase in the number of rapid (in the SVZ and the DG) and slow (in the SVZ) proliferating cells. Cumulative assays revealed a global reduction of the total cell cycle duration of rapidly proliferating precursors of SVZ. We investigated the fate of supernumerary cells and observed an increased number of apoptotic cells (terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling) in the mutant SVZ. Furthermore, we found no difference in the size of the olfactory bulb between wild-type (WT) and mutant mice. In support, mCD24 deletion did not appear to affect migration in the migratory stream. A comparison of the organization of migrating precursors between WT and mCD24 -/-, both in vivo at the optic and electron microscopic levels and in SVZ cultured explants, did not show any changes in the arrangement of neuroblasts in chain-like structures. Altogether, our data suggest that mCD24 regulates negatively cell proliferation in zones of secondary neurogenesis.
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