Neural cell adhesion molecule stimulates survival of premyelinating oligodendrocytes via the fibroblast growth factor receptor.

Axonal signals are critical in promoting the survival and maturation of oligodendrocytes during myelination, with contact-dependent signals thought to play a key role. However, the exact nature of these signals remains unclear. Neural cell adhesion molecule (NCAM) is expressed by both axons and oligodendrocytes and is ideally localized to transduce signals from the axon. This study sought to investigate the influence of NCAM on premyelinating oligodendrocytes in vitro. Both a soluble molecule comprising the extracellular domain of NCAM and a peptide derived from the fibroblast growth factor receptor (FGFR) binding motif within the first fibronectin domain stimulated a dose-dependent increase in survival of premyelinating oligodendrocytes in vitro. The survival effect was blocked by a mitogen-activated protein kinase (MAPK) inhibitor and an FGFR inhibitor, suggesting that activation of MAPK signalling pathways following interaction with the FGFR is involved in the survival effect of NCAM. Furthermore, NCAM presented in a cellular monolayer induced an increase in radial process outgrowth of oligodendrocyte progenitor cells. These data suggest that NCAM may play a role in axon-oligodendrocyte signalling during myelination, leading to an increase in oligodendrocyte survival and process outgrowth following axonal contact.

Morphoregulation by acetylcholinesterase in fibroblasts and astrocytes.

Acetylcholinesterase (AChE) terminates neurotransmission at cholinergic synapses by hydrolysing acetylcholine, but also has non-enzymatic morphoregulatory effects on neurons such as stimulation of neurite outgrowth. It is widely expressed outside the nervous system, but its function in non-neuronal cells is unclear. Here we have investigated the distribution and function of AChE in fibroblasts and astrocytes. We show that these cells express high levels of AChE protein that co-migrates with recombinant AChE but contains little catalytic activity. Fibroblasts express transcripts encoding the synaptic AChE-T isoform and its membrane anchoring peptide PRiMA-I. AChE is strikingly distributed in arcs, rings and patches at the leading edge of spreading and migrating fibroblasts and astrocytes, close to the cell-substratum interface, and in neuronal growth cones. During in vivo healing of mouse skin, AChE becomes highly expressed in re-epithelialising epidermal keratinocytes 1 day after wounding. AChE appears to be functionally important for polarised cell migration, since an AChE antibody reduces substratum adhesion of fibroblasts, and slows wound healing in vitro as effectively as a beta1-integrin antibody. Moreover, elevation of AChE expression increases fibroblast wound healing independently of catalytic activity. Interestingly, AChE surface patches precisely co-localise with amyloid precursor protein and the extracellular matrix protein perlecan, but not focal adhesions or alpha-dystroglycan, and contain a high concentration of tyrosine phosphorylated proteins in spreading cells. These findings suggest that cell surface AChE, possibly in a novel signalling complex containing APP and perlecan, contributes to a generalised mechanism for polarised membrane protrusion and migration in all adherent cells.

Structure of the tandem fibronectin type 3 domains of neural cell adhesion molecule.

Activation of the fibroblast growth factor receptor (FGFR) by neural cell adhesion molecule (NCAM) is essential for NCAM-mediated neurite outgrowth. Previous peptide studies have identified two regions in the fibronectin type 3 (FN3)-like domains of NCAM as being important for these activities. Here we report the crystal structure of the NCAM FN3 domain tandem, which reveals an acutely bent domain arrangement. Mutation of a non-conserved surface residue (M610R) led to a second crystal form showing a substantially different conformation. Thus, the FN3 domain linker is highly flexible, suggesting that it corresponds to the hinge seen in electron micrographs of NCAM. The two putative FGFR1-binding segments, one in each NCAM FN3 domain, are situated close to the domain interface. They form a contiguous patch in the more severely bent conformation but become separated upon straightening of the FN3 tandem, suggesting that conformational changes within NCAM may modulate FGFR1 activation. Surface plasmon resonance experiments demonstrated only a very weak interaction between the NCAM FN3 tandem and soluble FGFR1 proteins expressed in mammalian cells (dissociation constant >100 muM). Thus, the NCAM-FGFR1 interaction at the cell surface is likely to depend upon avidity effects due to receptor clustering.

Ligand concentration is a driver of divergent signaling and pleiotropic cellular responses to FGF.

Fibroblast growth factors (FGFs) are soluble ligands important for embryonic patterning, limb and brain development, and stem cell proliferation. They activate specific receptors (FGFR) to elicit changes in gene expression and cellular responses such as proliferation, differentiation, and survival, but the extent to which these pleiotropic responses are driven by FGF concentration gradients has not been systematically addressed. Here, we show that a single cell type exhibits divergent, even opposing, responses to a single FGF dependent on the exposure concentration, and that this is controlled by differential signaling with specific negative feedback inhibition. Low concentrations of FGF2 stimulate survival and differentiation but actively inhibit proliferation while intermediate concentrations stimulate proliferation in the presence of serum but apoptosis in its absence. Intriguingly, high concentrations reverse the proliferation and apoptosis effects, and mirror the low concentration effects: inhibition of proliferation and stimulation of survival and differentiation. By screening for activation of sampled signaling intermediates across the FGF2 concentration range in fibroblasts, we show that the peak in proliferation and apoptosis correlates with abrupt activation of FRS-2 and Erk that is specifically down-regulated by high concentrations of FGF2, a pattern that contrasts with an incremental increase in activation of p38 MAP kinase and the FGFR itself, across the FGF2 concentration range. Whilst proliferation stimulated by FGF2 was dependent on p38 MAP kinase, apoptosis stimulated by proliferative concentrations of FGF2 under serum-free conditions was, in contrast, dependent on Erk MAP kinase. These findings indicate that FGF exposure concentration precisely controls intracellular signaling and cellular responses to the growth factor, and have important implications for understanding how FGF gradients influence cell proliferation, survival, and differentiation during processes such as limb development.

A peptide from the first fibronectin domain of NCAM acts as an inverse agonist and stimulates FGF receptor activation, neurite outgrowth and survival.

Neural cell adhesion molecule (NCAM) contributes to axon growth and guidance during development and learning and memory in adulthood. Although the Ig domains mediate homophilic binding, outgrowth activity localizes to two membrane proximal fibronectin-like domains. The first of these contains a site identified as a potential FGF receptor (FGFR) activation motif (FRM) important for NCAM stimulation of neurite outgrowth, but its activity has hitherto remained hypothetical. Here, we have tested the effects of a domain-specific antibody and peptides corresponding to the FRM in cellular assays in vitro. The first fibronectin domain antibody inhibited NCAM-stimulated outgrowth, indicating the importance of the domain for NCAM function. Monomeric FRM peptide behaved as an inverse agonist; low concentrations specifically inhibited neurite outgrowth stimulated by NCAM and cellular responses to FGF2, while saturating concentrations stimulated FGFR-dependent neurite outgrowth equivalent to NCAM itself. Dendrimeric FRM peptide was 125-fold more active and stimulated FGFR activation, FGFR-dependent and FGF-mimetic neurite outgrowth and cell survival (but not proliferation). We conclude that the FRM peptide contains NCAM-mimetic bioactivity accounted for by stimulation of FGF signalling pathways at the level of or upstream from FGF receptors, and discuss the possibility that FRM comprises part of an FGFR activation site on NCAM.