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Biomedical subjects

F S Walsh

Publications and source records attributed to F S Walsh.

At least 73 records · Page 4Linked to original sources
PubMed1995

A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMs and FGF.

We have used monolayers of parental 3T3 fibroblasts and 3T3 cells expressing transfected cell adhesion molecules (CAMs, NCAM, N-cadherin, or L1) as a culture substrate for cerebellar neurons. Previous studies suggest that the transfected CAMs promote neurite outgrowth by activating a second messenger pathway within the responding neuron that involves influx of calcium into neurons as a consequence of activation of an FGF receptor. The same neurite outgrowth response can be induced by FGF or a number of agents that directly activate defined steps in the CAM signaling pathway. In the present study we show that the neurite outgrowth stimulated by the above three CAMs, FGF, arachidonic acid (AA), and K+ depolarization can be abolished by the Ca2+/calmodulin-dependent (CaM) kinase inhibitor, KN-62. We also demonstrate that neurite outgrowth over astrocytes, which represent a more physiologically relevant cellular substrate, can be substantially inhibited by a number of agents that block the CAM signaling pathway, including KN-62. However, neurite outgrowth induced by activation of protein kinase A is unaffected by inhibition of CaM kinase activity as is basal neurite outgrowth over 3T3 monolayers or a polylysine/laminin substrate. These results suggest that CaM kinase activity is specifically required downstream of calcium influx in the CAM and FGF signaling pathway leading to axonal growth.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

A soluble chimeric form of the L1 glycoprotein stimulates neurite outgrowth.

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/extracellular matrix-coated substratum, responded to a soluble recombinant L1-Fc chimera by extending longer neurites than controls. The response was inhibited by pretreating neurons with antibodies to L1 or antibodies to the fibroblast growth factor (FGF) receptor. The response could also be inhibited by a range of pharmacological reagents that inhibit various steps in the signal transduction cascade which underlie a neurite outgrowth response to basic FGF. The response was of a similar magnitude and not additive with that induced by L1 expressed in a cellular substrate. These data show that L1 in neurons is capable of directing a neurite outgrowth response to a soluble L1-Fc chimera, and that neuronal FGF receptor function is required for this response. The data also show that the ability of cell adhesion molecules (CAMs) to stimulate neurite outgrowth can be dissociated from their ability to function as substrate-associated adhesion molecules and point to the potential of using CAM-Fc chimeras to promote nerve regeneration.

3T3 Cells↗
PubMed1995

Efficiency of in vivo gene transfer using murine retroviral vectors is strain-dependent in mice.

Retroviral vectors can be used to transduce cultured cells at high frequencies, but efficient transduction of target cells in vivo has proved difficult and little is known about the factors that influence the efficiency of retroviral infection. Many commonly used mouse strains harbor endogenous C-type proviruses, some of which are expressed and have circulating antibodies against the viral envelope glycoproteins that cross-react with the Moloney strain of murine leukemia virus (MoMLV), from which most current retroviral vectors are derived. We have investigated the relative efficiency of retroviral-mediated gene transfer into regenerating skeletal muscle of a variety of mouse strains using a MoMLV-based vector. Humoral immune competence and interference between endogenous MLVs and exogenous recombinant MoMLV were observed to affect the efficiency of retroviral-mediated transfection in vivo. Our results indicate that the mouse genetic background and immune status need to be considered when choosing a preclinical model for in vivo retroviral-mediated gene transfer.

Animals↗
PubMed1995

Expression of human full-length and minidystrophin in transgenic mdx mice: implications for gene therapy of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder with a high spontaneous mutation rate and no effective treatment, hence development of genetic based therapies is an important goal. We report that expression of a recombinant human minidystrophin cDNA, compatible with current viral vectors, can significantly reduce the myopathic phenotype in transgenic mdx mice, even when expressed at only 20-30% of endogenous dystrophin levels at the sarcolemma. To the extent that data obtained in mouse studies are applicable to DMD, the virtual elimination of morphological and biochemical abnormalities in the mdx mouse supports the use of this cDNA in somatic gene therapy protocols for DMD.

Animals↗
PubMed1995

FGF inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules.

We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules.

3T3 Cells↗
PubMed1994

Transfection of glioma cells with the neural-cell adhesion molecule NCAM: effect on glioma-cell invasion and growth in vivo.

The tumor growth and the invasive capacity of a rat glioma cell line (BT4Cn) were studied after transfection with the human transmembrane 140-kDa isoform of the neural-cell adhesion molecule, NCAM. After s.c. injection, the NCAM-transfected cells showed a slower growth rate than the parent cell line (BT4Cn). Upon intracerebral implantation with BT4Cn cells and different clones of NCAM-transfected cells, all animals developed neurological symptoms within 13-16 days. However, the tumors showed different growth characteristics. The NCAM-transfected BT4Cn cells were localized in the region of the injection site, with a sharply demarcated border between the tumor and brain tissue. In contrast, the parental cell line showed single-cell infiltration and more pronounced destruction of normal brain tissue. Using a 51Cr-release assay, spleen cells from rats transplanted with BT4Cn tumor cells generally showed a lower cytotoxic response than the spleen cells from rats transplanted with the transfected variants of BT4Cn cells, indicating that the transfection procedure in itself mediated an activation of the immune system. The present data suggest that NCAM may influence the malignant behavior of rat glioma cells in vivo.

Animals↗
PubMed1994

Structure of the human N-cadherin gene: YAC analysis and fine chromosomal mapping to 18q11.2.

The cadherins are a large family of cell adhesion molecules involved in calcium-dependent recognition and adhesion events. We have used YAC analysis to determine the structure of the human N-cadherin gene. An 850-kb YAC was isolated and the entire N-cadherin gene mapped to a 250-kb region spanning three putative CpG islands. A PCR and cosmid subcloning strategy was used to define the boundaries for the 16 exons that compose the gene. These were shown to be not only highly conserved between mouse and human N-cadherin genes, but also similar to other cadherins. The first and second introns of the gene are large, a property conserved between the mouse and human genes. In situ hybridization with YAC DNA refined the map position of N-cadherin to human chromosome 18q11.2.

Animals↗
PubMed1994

Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin.

Cell contact-dependent neurite outgrowth stimulated by CAMs requires activation of a second messenger pathway that requires the function of a tyrosine kinase upstream from calcium influx into neurons. In the present study, we present evidence that implicates activation of the fibroblast growth factor receptor (FGFR) in the pathway underlying neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. We have identified a CAM homology domain in the FGF family of receptors and show that antibodies which bind to this domain specifically inhibit neurite outgrowth stimulated by the above CAMs. We also show that synthetic peptides derived from this domain can differentially and specifically inhibit neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. In addition, a soluble L1-Fc chimera is shown to stimulate an increase in phosphotyrosine on the same set of neuronal proteins that are phosphorylated following activation of the FGFR with basic FGF.

Amino Acid Sequence↗
PubMed1994

A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration.

Following nerve injury, axons in the CNS do not normally regenerate. It has been shown that CNS myelin inhibits neurite outgrowth, though the nature of the molecules responsible for this effect are not known. Here, we demonstrate that the myelin-associated glycoprotein (MAG), a transmembrane protein of both CNS and PNS myelin, strongly inhibits neurite outgrowth from both developing cerebellar and adult dorsal root ganglion (DRG) neurons in vitro. This inhibition is reversed by an anti-MAG antibody. In contrast, MAG promotes neurite outgrowth from newborn DRG neurons. These results suggest that MAG may be responsible, in part, for the lack of CNS nerve regeneration in vivo and may influence, both temporally and spatially, regeneration in the PNS.

Animals↗
PubMed1994

Signal transduction events underlying neurite outgrowth stimulated by cell adhesion molecules.

During development of the nervous system, cell adhesion molecules (CAMs) promote cell migration and axonal growth; yet, at other times, CAMs inhibit these events by maintaining stable adhesion between cells. In the present review, we consider recent results that help to explain the paradoxical findings that individual CAMs can both promote and inhibit neuronal plasticity. In particular, we discuss the accumulating evidence that axonal growth stimulated by CAMs depends upon the activation of a second messenger pathway that culminates in calcium entry into neurons rather than on adhesion per se.

Animals↗
PubMed1994

The production of arachidonic acid can account for calcium channel activation in the second messenger pathway underlying neurite outgrowth stimulated by NCAM, N-cadherin, and L1.

We have used monolayers of control 3T3 fibroblasts and 3T3 fibroblasts expressing transfected cell adhesion molecules (CAMs)--NCAM, N-cadherin, and L1--as a culture substrate for cerebellar neurones. The transfected CAMs promote neurite outgrowth by activating a second messenger pathway that culminates in calcium influx into neurones through N- and L-type calcium channels. We show that the same neurite outgrowth response can be directly induced by arachidonic acid (10 microM) and that this response can be inhibited by N- and L-type calcium channel antagonists. In cells, arachidonic acid can be generated by phospholipase A2 or by the sequential activities of a phospholipase C (to generate diacylglycerol) and diacylglycerol lipase. In the present study we show the neurite outgrowth stimulated by CAMs (but not by various other agents) can be abolished by an inhibitor of diacylglycerol lipase acting at a site upstream from calcium channel activation. The results suggest that arachidonic acid and/or one of its metabolites is the second messenger that activates calcium channels in the CAM signalling pathway leading to axonal growth, and this is supported by recent evidence that shows the same concentrations of arachidonic acid can increase voltage-dependent calcium currents in cardiac myocytes.

3T3 Cells↗
PubMed1994

Neurite outgrowth stimulated by the tyrosine kinase inhibitor herbimycin A requires activation of tyrosine kinases and protein kinase C.

Activation of tyrosine kinases is established as an important mechanism for controlling growth cone motility and neurite outgrowth. We have tested the effects of a range of tyrosine kinase inhibitors on neurite outgrowth from postnatal day 4 cerebellar granule cells cultured over confluent monolayers of 3T3 fibroblasts. The only agent that had any effect was herbimycin A, which stimulated neurite outgrowth. The response is shown to be attributable to a direct effect of this tyrosine kinase inhibitor on neurones. The neurite outgrowth response to herbimycin A was inhibited by two other tyrosine kinase inhibitors, which on their own did not affect neurite outgrowth. The data suggest that the response to herbimycin A reflects either a direct or indirect activation of one or more protein tyrosine kinases. Independent signalling events down-stream from tyrosine kinase activation underlying the neurite outgrowth response to herbimycin A include increased activity of protein kinase C and calcium influx into neurones through both N- and L-type calcium channels.

Animals↗
PubMed1994

Tyrosine kinase inhibitors can differentially inhibit integrin-dependent and CAM-stimulated neurite outgrowth.

We have used monolayers of parental 3T3 cells and 3T3 cells expressing one of three transfected cell adhesion molecules (CAMs) (NCAM, N-cadherin, and L1) as a culture substrate for rat cerebellar neurons. A number of tyrosine kinase inhibitors have been tested for their ability to inhibit neurite outgrowth over parental 3T3 monolayers which we show to be partly dependent on neuronal integrin receptor function, as compared with neurite outgrowth stimulated by the above three CAMs. Whereas genistein (100 microM), lavendustin A (20 microM), and tyrphostins 34 and 47 (both at 150 microM) had no effect on integrin dependent or CAM stimulated neurite outgrowth, the erbstatin analogue (10-15 micrograms/ml) and tyrphostins 23 and 25 (both at 150 microM) specifically inhibited the response stimulated by all three CAMs. CAM stimulated neurite outgrowth can be accounted for by a G-protein-dependent activation of neuronal calcium channels; experiments with agents that directly activate this pathway localized the erbstatin analogue site of action upstream of the G-protein and calcium channels, whereas tyrphostins have sites of action downstream from calcium channel activation. These data suggest that activation of an erbstatin sensitive tyrosine kinase is an important step upstream of calcium channel activation in the second messenger pathway underlying the neurite outgrowth response stimulated by a variety of CAMs, and that this kinase is not required for integrin-dependent neurite outgrowth.

3T3 Cells↗
PubMed1994

Expression of NCAM containing VASE in neurons can account for a developmental loss in their neurite outgrowth response to NCAM in a cellular substratum.

Binding of the neural cell adhesion molecule (NCAM) in neurons to NCAM on non-neuronal cells can stimulate axonal growth. A developmentally regulated loss of this response is associated with the insertion of 10 amino acids (called VASE) into the fourth Ig domain in up to 50% of the NCAM receptors in neurons. In the present study we have transfected PC12 cells with the major neuronal isoforms of human NCAM and tested cells expressing these isoforms for their ability to respond to NCAM in a cellular substratum. Whereas both the 140- and 180-kD isoforms of NCAM can act as functional receptors for neurite outgrowth, the presence of the VASE sequence in a minority of the receptors specifically inhibited this response. A synthetic peptide containing the VASE sequence inhibits neurite outgrowth from PC12 cells and primary neurons stimulated by NCAM. The same peptide has no effect on integrin dependent neurite outgrowth or neurite outgrowth stimulated by N-cadherin or L1. We discuss the possibility that the VASE peptide inhibits the NCAM response by preventing NCAM from binding to the FGF receptor in the plasma membrane.

3T3 Cells↗
PubMed1994

Characterisation of the second messenger pathway underlying neurite outgrowth stimulated by FGF.

Cerebellar neurons, cultured on monolayers of 3T3 fibroblasts or on a polylysine/laminin-coated substratum, responded to recombinant basic FGF by extending longer neurites. The response was biphasic reaching a maximum at 5 ng/ml FGF, but desensitising at 100-200 ng/ml FGF. The response to FGF could be inhibited by a tyrosine kinase inhibitor (the erbstatin analogue), by a diacylglycerol lipase inhibitor (RHC-80267) and by a combination of N- and L-type calcium channel antagonists or other agents that negate the effects of calcium influx into neurons. The response to FGF could be fully mimicked by arachidonic acid added directly to the cultures, or generated via activation of phospholipase A2 with melittin. The response to melittin, but not to FGF or arachidonic acid, was inhibited by 4-bromophenacyl bromide, a phospholipase A2 inhibitor. The response to arachidonic acid was also biphasic and high concentrations of this agent could cross-desensitise the FGF response and vice versa. The response to arachidonic acid could be fully inhibited by the agents that block or negate the effects of calcium influx into neurons, but was not inhibited by the tyrosine kinase or diacylglycerol lipase inhibitors. These data suggest that FGF stimulates neurite outgrowth by activating a cascade that involves activation of phospholipase C gamma to produce diacylglycerol, conversion of diacylglycerol to arachidonic acid by diacylglycerol lipase and the activation of voltage-gated calcium channels by arachidonic acid.

3T3 Cells↗
PubMed1994

Neurite outgrowth stimulated by L1 requires calcium influx into neurons but is not associated with changes in steady state levels of calcium in growth cones.

L1, NCAM and N-cadherin are cell adhesion molecules (CAMs), present on neuronal growth cones, which promote cell-contact dependent axonal growth by activating a second messenger pathway in neurons that requires calcium influx through L- and N-type calcium channels. In the present study we show that two of these CAMs, (L1 and N-cadherin) can stimulate neurite regeneration from axotomised adult dorsal root ganglion (DRG) neurons cultured in vitro and that this response can be fully inhibited by agents that block or negate the effect of calcium influx into the neurons. However although the response required calcium influx into neurons, it was not associated with an increase in the steady state levels of calcium in neuronal growth cones. These results suggest that small localised changes, or increases in the rate of calcium cycling, in growth cones and/or filopodia, are more important for regulating axonal growth than changes in the steady-state level of calcium.

3T3 Cells↗