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F S Walsh

Publications and source records attributed to F S Walsh.

At least 55 records · Page 3Linked to original sources
PubMed1997

Cell signalling and CAM-mediated neurite outgrowth.

A wide range of molecules promote nerve growth, and these include cell adhesion molecules (CAMs), NCAM, N-cadherin, and the L1 glycoprotein are CAMs that are normally found on both the advancing growth cone and also on cellular substrates, and in general operate via a homophilic binding mechanism. In recent years it has become clear that nerve growth stimulated by these CAMs does not rely on the adhesion function of these molecules, but instead requires that the CAMs activate second messenger cascades in neurons. A large body of evidence supports the hypothesis that homophilic binding of the CAM in the substrate to the CAM in the neuron leads to activation of the neuronal FGF receptor, possibly via a direct interaction in cis between the CAM and the FGF receptor. The consequential activation of PLC gamma is both necessary and sufficient to account for the neurite outgrowth response stimulated by the above three CAMs. Based on the above model, we reasoned that soluble CAMs might also be able to stimulate neurite outgrowth and that such agents might be developed as potential therapeutic agents for stimulating nerve regeneration. To this end we have made soluble chimeric molecules consisting of the extracellular domain of NCAM or L1 fused to the Fc region of human IgG 1. We have found that these molecules can stimulate neurite outgrowth from rat and mouse cerebellar granule cells cultured on a variety of tissue culture substrates and that they do so by activating the FGF receptor signal transduction cascade in the neurons. Consistent with this model, we find that neurons that have their FGF receptor function ablated as a consequence of the expression of dominant negative FGF receptors, no longer respond to the soluble CAM. Downstream targets of CAM function have also been studied. Addition of soluble CAMs to isolated growth cone preparations from mouse or rat brain leads to enhanced phosphorylation of the GAP-43 protein providing a link between the cell surface and the cytoskeleton.

Animals↗
PubMed1997

Signal transduction mechanisms underlying axonal growth responses stimulated by cell adhesion molecules.

The mechanisms underlying nerve growth have been extensively studied, and it has been found that the three cell adhesion molecules (CAMs) L1, NCAM and N-cadherin play a key role in this process. All three CAMs are able to stimulate axonal growth from a variety of neuronal cells, and a range of agents which either mimic or inhibit CAM stimulated neurite outgrowth have been identified and has provided a basis for understanding the nature of the response. Results from these studies suggested that activation of a tyrosine kinase-phospholipase C gamma (PLC gamma) cascade was required for the CAM response. Following the identification of a CAM-homology domain (CHD) within the fibroblast growth factor receptor (FGFR) and a putative CHD-binding motif within each of the CAMs, it was suggested that this might be the tyrosine kinase implicated in the CAM pathway. This has been tested experimentally in a number of ways, including the use of transgenic mice expressing a dominant-negative FGFR, and several results have now demonstrated that a functional FGFR is required for CAM stimulated neurite outgrowth. More recently, treatment of neurons with the CAMs has been shown to stimulate FGFR autophosphorylation and PLC gamma activity which in turn leads to activation of a second messenger cascade involving diacylglycerol and arachidonic acid and results in calcium influx into the neurons. Pharmacological studies have confirmed that this cascade is responsible for the neurite outgrowth response.

Animals↗
PubMed1996

The p53 response to ionising radiation in adult and developing murine tissues.

The induction of the p53 response to ionising radiation has been studied during murine development and in the adult animal. The response has been assessed by precise quantitative assay of p53 protein levels in tissues and by immunohistochemistry. Newly developed transgenic mice in which a lacZ transgene is driven by a p53 response element have also been used to directly assess the transcriptional activity of the induced protein. There is striking developmental control of the p53 response so that in early development all tissues accumulate high levels of p53 following radiation and indeed p53 is present at elevated levels in some unirradiated tissues. Later in development clear heterogeneity of the p53 response becomes apparent, both in terms of the responses of individual tissues and of cell populations, within those tissues. The study of lacZ transgene expression and the occurrence of apoptosis in different tissues that accumulate p53 protein point to a further level of control regulating the nature and degree of the downstream response to elevated levels of p53 in cells. These findings have important implications for the susceptibility of different tissue types to carcinogenic and other insults. The early expression of the p53 response is consistent with novel models of p53 function that suggest it may have evolved principally as a defense against teratogenic insult that permits plasticity of development.

Age Factors↗
PubMed1996

Elucidation of the molecular actions of NCAM and structurally related cell adhesion molecules.

The Neural Cell Adhesion Molecule (NCAM) is a founder member of a large family of cell surface glycoproteins that share structural motifs related to immunoglobulin and fibronectin type III (FN III) domains [Walsh and Doherty (1991) (Fig. 1). These glycoproteins have been grouped based on the respective number of each type of domain. In vertebrates members of this family of glycoproteins include L1/NILE, NgCAM, axonin-1/TAG-1, and Thy-1 as well as NCAM. In addition structural homologs of NCAM and L1 have been identified in Drosophila and Grasshoppers [Walsh and Doherty (1991)]. These insect homologs are called fasciclins and a series of mutants corresponding to these aspects of synaptic plasticity [Mayford et al. (1992) Science 256:638-644]. In vertebrates all of these glycoproteins are expressed in the developing nervous system where they have been identified as candidate molecules for mediating axon outgrowth, fasciculation, regeneration, and target recognition. In addition, NCAM is expressed in a number of different tissues and cell types. For example, NCAM is expressed in a dynamic pattern in developing and regenerating adult muscle. In this review we aim to describe important aspects of the role of these CAMS in development of the nervous system, including the neuromuscular junction. Furthermore, we will explore the prospective use of molecular biology, cell biology, and molecular genetic techniques, such as transgenic mice, to understand the role and molecular action of this family of cell adhesion molecules in vivo.

Amino Acid Sequence↗
PubMed1996

Inhibition of FGF-stimulated phosphatidylinositol hydrolysis and neurite outgrowth by a cell-membrane permeable phosphopeptide.

BACKGROUND: Activated receptor tyrosine kinases bind downstream effector molecules with high affinity. Provided that they can be introduced into cells, peptides corresponding to these high-affinity sites should be able to compete for the interaction and thereby inhibit specific signal transduction cascades. The high-affinity binding site for phospholipase C gamma (PLCgamma) on the activated fibroblast growth factor receptor (FGFR) is centred around the tyrosine at position 766 (766Tyr), and peptides corresponding to this site inhibit PLCgamma binding to the receptor in vitro. A 16 amino-acid peptide from the third helix of the Antennapedia homeodomain protein has recently been shown to be able to act as an internalization vector that can deliver other peptides into cells. Here, we have designed a peptide that contains both the internalization sequence and the FGFR high-affinity binding site for PLCgamma, and tested it in cultures of cerebellar neurons for its ability to inhibit the activation of PLCgamma by basic FGF. RESULTS: The peptide containing the FGFR high-affinity binding site for PLCgamma inhibited phospholipid hydrolysis stimulated by basic FGF with a maximal effect at 1 microg ml-1. Phosphorylation of 766Tyr was required for this effect. The phosphorylated peptide had no effect on phospholipid hydrolysis stimulated by platelet-derived growth factor, neurotrophin-3 and bradykinin. The phosphorylated peptide also inhibited neurite outgrowth stimulated by FGF, but had no effect on neurite outgrowth stimulated by agents that activate the FGFR signal transduction cascade downstream from the activation of PLCgamma. CONCLUSIONS: Cell-permeable peptides can be designed that inhibit the function of receptor tyrosine kinases. In this context we have developed a peptide that prevents the FGFR from activating PLCgamma, and have used this peptide to obtain the first direct evidence that activation of PLCgamma is required for the neurite outgrowth response stimulated by basic FGF.

Amino Acid Sequence↗
PubMed1996

Transplantation of retroviral producer cells for in vivo gene transfer into mouse skeletal muscle.

We describe a new strategy for efficient in vivo gene transfer into skeletal muscle using retroviral vectors. Recombinant retroviral producer cells, previously treated with the cytostatic drug mitomycin C, were injected into regenerating muscle of adult nude, nude/mdx, and C57BL/10 mice. Using LacZ reporter gene activity, we detected efficient transduction in all mouse strains (Nude, mean 11%, range 4.2-21%; C57BL/10, mean 12%, range 3.4-20%; Nude mdx, mean 4.3%, range 2.1-7% at 4 weeks post-injection and 6.6%, range 1.3-12% at 12 weeks post-injection). Foreign gene expression was sustained at high levels for at least 3 months. This strategy allows muscle satellite cells to be transfected in vivo, forming a reservoir of the transgene for incorporation into new myofibers in subsequent rounds of degeneration and regeneration. Because of its efficiency and potentially broad application, this procedure represents a new strategy for in vivo genetic transfer in skeletal muscle and potentially in other tissues.

Animals↗
PubMed1996

Promiscuity of fibroblast growth factor receptors.

Fibroblast growth factor receptors (FGFRs) have been implicated in many developmental and regenerative events, including axial organisation, mesodermal patterning, keratinocyte organisation and brain development. The consensus view that this reflects a role for one or other of the nine known members of the fibroblast growth factor family in these processes has recently been challenged by the suggestion that FGFRs might be directly activated by a much wider range of ligands, including heparan sulphate proteoglycans and neural cell adhesion molecules. In addition, two novel soluble ligands for FGFRs have been identified using yeast two-hybrid technology. Overall, the new findings suggest that in terms of ligand binding the FGFRs might be an even more promiscuous family of receptor tyrosine kinases than was already appreciated.

Amino Acid Sequence↗
PubMed1996

CAM-FGF receptor interactions: a model for axonal growth.

A number of experimental paradigms have been used to demonstrate that NCAM, N-cadherin, and L1 stimulate axonal growth. The molecular basis of this response has been extensively studied and a range of agents that inhibit neurite outgrowth stimulated by the above CAMs, but not integrins, have now been identified. These studies pointed to the activation of a tyrosine kinase-PLCgamma cascade as being important for the neurite outgrowth responses stimulated by all three CAMs, and this was substantiated by the identification of agents that could activate the cascade and mimic the growth response. In this review we will suggest that the neurite growth response stimulated by these CAMs is mediated by activation of the fibroblast growth factor receptor (FGFR) in neurons and that this results in the recruitment and activation of PLCgamma via interactions of its SH2 domain with the activated receptor. In this context the key events downstream from activation of PLCgamma required for neurite growth appear to be the conversion of diacylglycerol (DAG) to arachidonic acid (AA) via DAG lipase activity, followed by an increased influx of calcium into the neurons. The evolutionary conservation of putative binding motifs between the above CAMs and the FGFR suggests that activation of the FGFR-PLCgamma cascade by the CAMs might involve a direct CAM-FGFR interaction. The identification of the binding motifs also allows for predictions to be made concerning whether other CAMs might directly interact with the FGFR.

Amino Acid Sequence↗
PubMed1996

Fibroblast growth factor receptor function is required for the orderly projection of ganglion cell axons in the developing mammalian retina.

During the early stages of development various cell adhesion molecules (CAMs) and fibroblast growth factor receptors (FGFR) are expressed throughout the retinal neuroepithelium. The ability of retinal ganglion cells to project their axons to the optic fissure depends, in part, on cell-cell interactions mediated by cell adhesion molecules. In the present study we show that the ability of the firstborn rat retinal ganglion cells to extend axons in vitro can be stimulated by NCAM and L1, but not N-cadherin. Both CAM responses can be fully inhibited by antibodies that block neuronal fibroblast growth factor receptor function and by agents that block defined steps in the FGFR signal transduction cascade. When added to living E13.5 rat retinal whole-mount preparations the same agents induced errors in the orderly establishment of young axon patterns in the retinal periphery and caused axons in the retinal center to defasciculate. These results suggest that the activation of the fibroblast growth factor receptor signal cascade not only promotes survival and proliferation of various cell types but can also mediate intraretinal axon guidance.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1996

Idiopathic constipation is not associated with increased NCAM expression on intestinal muscle.

Our previous study of neural cell adhesion molecule (NCAM) in childhood Hirschsprung's disease demonstrated increased expression on intestinal muscle, especially muscularis mucosae, in aganglionic bowel. The present study was undertaken to test whether this increased NCAM expression is a feature of congenital aganglionosis rather than being a nonspecific feature of constipation. We studied specimens from six patients (20-60 years old; five women and one man) operated on for idiopathic long-standing constipation, using immunocytochemistry for NCAM and protein gene product 9.5 (PGP 9.5). Results showed that in adult constipated bowel the pattern of NCAM expression is similar to that seen in controls. There was no expression of NCAM on muscle in any of the specimens studied. Thus, it seems likely that increased NCAM expression in gastrointestinal smooth muscle in congenital aganglionosis is a result of the abnormal innervation rather than any symptomatic or clinical feature.

Adult↗
PubMed1996

Cell adhesion molecules and neuronal regeneration.

Cell adhesion molecules (CAMs) are multifunctional proteins and are involved in a number of important regulatory processes in the brain, including cell growth, migration and regeneration. Recent studies using model in vitro systems have identified additional binding interactions in which CAMs, particularly those of the Ig superfamily, can participate. Signal transduction pathways are activated following CAM action in the process of neurite outgrowth. Key components in these pathways, such as kinases and phosphatases, are being identified. Receptor phosphatases themselves contain protein motifs characteristic of CAMs and may themselves be involved in adhesion-mediated cell recognition events.

Animals↗
PubMed1996

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter.

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol-anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.

Actins↗
PubMed1996

Neural cell adhesion molecule (NCAM) expression in nerves and muscle of developing human large bowel.

Most studies of neural cell adhesion molecule (NCAM) in human musculature are devoted to either developing or adult skeletal and cardiac muscle. The aim of this study was to determine the pattern of NCAM expression in the intestinal musculature of the developing human large bowel. In specimens of large bowel from foetuses (gestational age 8-20 weeks), we examined the immunohistochemical localisation of NCAM in parallel to those of alpha-smooth muscle actin and desmin. Within the developing neural complex, NCAM was expressed at all stages investigated. In intestinal muscle at 8 weeks, immunoreactivity for all antisera was restricted to the muscularis propria. The differentiating muscularis mucosae was demonstrated first at 15 weeks by immunostaining for alpha-smooth muscle actin, and this expression was followed by that of NCAM and desmin at 17 and 19 weeks, respectively. At 20 weeks, NCAM immunoreactivity in the external muscle was intense at the inner border of the circular muscle, with its concentration decreasing towards the outer margin of the muscular wall, whereas alpha-smooth muscle actin and desmin were uniformly distributed in all muscle layers. NCAM is expressed by nerves and muscle of developing human large intestine. Its appearance follows a predetermined pattern, which implies its relevance to the differentiation of intestinal muscle layers.

Actins↗
PubMed1996

Review: a role for the FGF receptor in the axonal growth response stimulated by cell adhesion molecules?

Cell adhesion molecules (CAMs) have been shown to stimulate axonal growth. The molecular basis of this response has been extensively studied and a range of agents that promote or inhibit CAM stimulated axonal growth have now been identified. These studies have led to the suggestion that following homophilic and/or heterophilic interactions CAM specific signal transduction pathways are activated which are directly responsible for promotion of axonal growth. In this review we will suggest that the axonal growth response stimulated by three CAMs (NCAM, N-cadherin and L1) can be operationally divided into a number of phases. During the first phase homophilic and/or heterophilic binding between the CAMs expressed on the axonal growth cone and cellular substrate take place. This is followed by an interaction of the neuronal CAMs with the fibroblast growth factor receptor (FGFR), leading to receptor activation by autophosphorylation. This results in the recruitment and activation of additional effector molecules via interactions of their SH2 domains with the activated receptor. In this context the key event in terms of neurite outgrowth appears to be the activation of phospholipase C gamma (PLC gamma) which sets into motion a second messenger cascade that ultimately leads to a modification, most likely by phosphorylation, of cytoskeletal elements that are involved in growth cone motility.

Animals↗
PubMed1996

Shared cell adhesion molecule (CAM) homology domains point to CAMs signalling via FGF receptors.

A number of cell adhesion molecules (CAMs) promote neurite outgrowth following transfection and expression in a variety of monolayer cells. We have shown that N-cadherin, L1 and some isoforms of NCAM can stimulate neurite outgrowth from PC12 cells and primary neurons following transfection and expression at physiologically relevant levels in NIH-3T3 cells. A number of observations suggest that these CAMs stimulate neurite outgrowth by activating a convergent second messenger pathway in neurons rather than by modulating adhesion per se, and that an early or initial step in the pathway involves activation of a tyrosine kinase. The observation that the fibroblast growth factor receptor (FGFR) contains an evolutionarily conserved sequence with homology to the above CAMs (the CAM homology domain-CHD) points to the possibility that CAMs might interact with, and signal via, FGFR tyrosine kinases. This hypothesis has been substantiated by a number of independent experimental tests. We present a speculative model in which the evolutionary conservation of a pair of complementary binding motifs can account for a direct binding interaction between FGFR and the above three CAMs.

Animals↗
PubMed1995

The neural cell adhesion molecule and synaptic plasticity.

Highly stereotyped patterns of neuronal connections are laid down during the development of the nervous system via a range of activity independent and activity dependent mechanisms. Whereas the coarse hard-wiring of the nervous system appears to rely on molecular recognition events between the neuron, its pathway, and its target, the establishment of precisely patterned functional circuits is thought to be driven by neuronal activity. In this review we discuss the role that the neuronal cell adhesion molecule (NCAM) plays in morphological plasticity. Recent studies on NCAM and its probable species homologue in Aplysia (apCAM) suggests that an individual CAM can function to both promote synaptic plasticity and maintain the structure of the synapse. In the adult brain, changes between stability and plasticity are likely to underlie dynamic morphological changes in synaptic structures associated with learning and memory. In this review we use NCAM as an example to illustrate mechanisms that can change the function of an individual CAM from a molecule that promotes plasticity to one that does not. We also discuss evidence that NCAM promotes plasticity by activating a conventional signal transduction cascade, rather than by modulating adhesion per se. Finally, we consider the evidence that supports a role for NCAM in learning and memory.

Animals↗
PubMed1995

NCAM requires a cytoplasmic domain to function as a neurite outgrowth-promoting neuronal receptor.

The neural cell adhesion molecule (NCAM) promotes axonal growth via a homophilic binding mechanism by acting both as a neuronal receptor and a substratum ligand. We have previously shown that the GPI-linked 120-kDa isoform of NCAM, which lacks a cytoplasmic domain, is effective at promoting neurite outgrowth as a cellular ligand. To test its ability to function as a neuronal receptor, we have transfected PC12 cells with a cDNA encoding human GPI-linked NCAM and tested clones displaying stable cell surface expression of this isoform for their ability to respond to NCAM in a cellular substratum. Although they continued to express endogenous transmembrane rat isoforms of NCAM (140 and 180 kDa), PC12 cells expressing the GPI-linked NCAM lost their ability to extend neurites in response to substratum associated NCAM. However, their outgrowth response to N-cadherin and other activators of axonal growth was undiminished. Removal of GPI-linked NCAM from the surface of these clones using phosphatidylinositol-specific phospholipase C (PIPLC) fully restored their responsiveness to NCAM, indicating that the inhibition was a direct consequence of cell surface expression of this "dominant negative" isoform of NCAM. We have previously shown that expression of transfected 140- and 180-kDa isoforms of human NCAM in PC12 cells does not result in a loss of the neurite outgrowth response to NCAM. However, we show that deletion of the cytoplasmic domain of the 140-kDa isoform has the same effect as expression of GPI-linked NCAM. We conclude that the cytoplasmic domain of NCAM is required for an appropriate neurite outgrowth response.

Animals↗