Biomedical subjects

F S Walsh

Publications and source records attributed to F S Walsh.

At least 37 records · Page 2Linked to original sources

PubMed1999

A soluble version of the receptor-like protein tyrosine phosphatase kappa stimulates neurite outgrowth via a Grb2/MEK1-dependent signaling cascade.

Receptor-like protein tyrosine phosphatase kappa (RPTPkappa) is expressed in the nervous system in a manner consistent with a role in axonal growth and guidance. The extracellular domain of RPTPkappa shares structural features with cell adhesion molecules and can support homophilic adhesion. In the present study we produced a soluble Fc-chimeric protein containing the full extracellular domain of RPTPkappa. Following affinity capture, the RPTPkappa-Fc was shown to promote the aggregation of Covasphere beads, confirming its homophilic binding activity. When added to cultures of cerebellar neurons as a soluble molecule, the RPTPkappa chimera stimulated neurite outgrowth. The neurite outgrowth response was substantially inhibited by a cell-permeable peptide inhibitor of Grb2 and by PD 098059, a drug that has been used to inhibit MEK1 activation in a wide range of cell types. These results demonstrate that RPTPkappa can stimulate neurite outgrowth and provide evidence that this might involve the coupling of Grb2 to a MAPK signal transduction cascade.

3T3 Cells↗

PubMed1999

Identification of a novel aspartic protease (Asp 2) as beta-secretase.

The Alzheimer's disease beta-amyloid peptide (Abeta) is produced by excision from the type 1 integral membrane glycoprotein amyloid precursor protein (APP) by the sequential actions of beta- and then gamma-secretases. Here we report that Asp 2, a novel transmembrane aspartic protease, has the key activities expected of beta-secretase. Transient expression of Asp 2 in cells expressing APP causes an increase in the secretion of the N-terminal fragment of APP and an increase in the cell-associated C-terminal beta-secretase APP fragment. Mutation of either of the putative catalytic aspartyl residues in Asp 2 abrogates the production of the fragments characteristic of cleavage at the beta-secretase site. The enzyme is present in normal and Alzheimer's disease (AD) brain and is also found in cell lines known to produce Abeta. Asp 2 localizes to the Golgi/endoplasmic reticulum in transfected cells and shows clear colocalization with APP in cells stably expressing the 751-amino-acid isoform of APP.

Alzheimer Disease↗

PubMed1999

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions.

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.

Animals↗

PubMed1998

Neurite outgrowth stimulated by neural cell adhesion molecules requires growth-associated protein-43 (GAP-43) function and is associated with GAP-43 phosphorylation in growth cones.

The mechanisms whereby cell adhesion molecules (CAMs) promote axonal growth and synaptic plasticity are poorly understood. Here we show that the neurite outgrowth stimulated by NCAM-mediated fibroblast growth factor (FGF) receptor activation in cerebellar granule cells is associated with increased GAP-43 phosphorylation on serine-41. In contrast, neither NCAM nor FGF was able to stimulate neurite outgrowth in similar neurons from mice in which the GAP-43 gene had been deleted by homologous recombination. Integrin-mediated neurite outgrowth was unaffected by GAP-43 deletion. Both neurite outgrowth and rapid phosphorylation of GAP-43 in isolated growth cones required the first three Ig domains of a NCAM-Fc chimera and were stimulated maximally at 5 micrograms/ml (approximately 50 nM). Likewise, GAP-43 phosphorylation in isolated growth cones also was stimulated by an L1-Fc chimera. Both neurite outgrowth and NCAM-stimulated GAP-43 phosphorylation were inhibited by antibodies to the FGF receptor and a diacylglycerol lipase inhibitor (RHC80267) that blocks the production of arachidonic acid in response to activation of the FGF receptor. Direct activation of the FGF receptor and the arachidonic acid cascade with either basic FGF or melittin also resulted in increased GAP-43 phosphorylation. These data suggest that the stimulation of neurite outgrowth by NCAM requires GAP-43 function and that GAP-43 phosphorylation in isolated growth cones occurs via an FGF receptor-dependent increase in arachidonic acid.

3T3 Cells↗

PubMed1998

Muscle-specific locus control region activity associated with the human desmin gene.

We describe the reproduction of the full pattern of expression of the muscle-specific desmin gene in transgenic mice using a 240-kb genomic clone spanning the human desmin locus. Analysis of RNA from adult tissues demonstrated that this fragment possesses all the necessary genetic regulatory elements required to provide reproducible, site-of-integration-independent, physiological levels of tissue-specific expression that is directly proportional to transgene copy number in all muscle cell types. In situ hybridization revealed that in marked contrast to murine desmin which is strongly expressed in the myotome of the somites, skeletal muscles, the heart, and smooth muscle of the vasculature by 9.5 days postcoitum, human desmin transgene expression was completely absent from smooth muscles, was very weak and restricted to the atrium and outflow tract within the heart, and was expressed at only 5% of murine desmin mRNA levels within the myotome of the somites. The spatial distribution and levels of human and mouse desmin expression were not coincident until 14.5 days postcoitum. Immunohistochemical analysis of human embryos at comparable stages of development showed that this transgene faithfully reproduces the human and not the mouse developmental expression pattern for this gene in transgenic mice. These results indicate that the 240-kb desmin genomic clone is capable of establishing an independent, chromatin domain in transgenic mice and provides the first definitive data for muscle-specific locus control region activity. In addition, our results demonstrate that the behavior of human transgenes in mice should, whenever possible, be compared to expression patterns for that gene in human embryonic as well as adult tissues.

Animals↗

PubMed1998

Migration of hypoglossal myoblast precursors.

The intrinsic hypoglossal musculature develops from precursor myoblasts which undergo long-range migration from the occipital somites to the tongue. Little detail is known about the precise spatiotemporal pathway taken by these cells or the factors controlling migration. In this study, chick/quail chimeras in which the occipital paraxial mesoderm is quail derived, reveal that the pathway taken by the tongue muscle progenitors is both complex and highly specific. Precursor myoblasts are Pax-3 positive cells which descend from the somite and migrate around the pharyngeal endoderm. They then course rostrally, following the base of the pharynx, remaining in a tight strand. We have examined a number of factors implicated in the control of migration of the hypoglossal precursors. Replacement of the occipital somites with those originating in the flank reveals that intrinsic differences do not exist between these somites with respect to their capacity to respond to migratory cues. The lack of high level HGF/SF expression along the pathway of the migrating hypoglossal precursors suggests that this factor is not involved in the actual process of migration of the hypoglossal precursors to the tongue. The pathway followed by the migrating precursors is identical to that of both the developing hypoglossal nerve and the circumpharyngeal crest--a subpopulation of the cranial neural crest, and importantly these populations utilize this pathway before the myoblast precursors. However, ablation neither of the hypoglossal nerve nor of the neural crest results in a perturbation in the ability of this Pax-3 positive population to migrate. This demonstrates that migration of the precursors is independent of both of these cell populations, and that it is controlled by the peripheral tissues.

Animals↗

PubMed1998

Neurotrophic molecules: strategies for designing effective therapeutic molecules in neurodegeneration.

Over the past several years, neurotrophic factors-a description generally applied to naturally occurring polypeptides that support the development and survival of neurons-have made considerable progress from the laboratory into the clinic. Evidence from preclinical and clinical studies indicates that it may be possible to use neurotrophic factors to prevent, slow the progression of, or even reverse the effects of a number of neurodegenerative diseases and other types of insults in both the central nervous system (CNS) and the peripheral nervous system. Initially, investigations focused on recombinant neurotrophic proteins that are identical or highly homologous to the natural human sequence. Given the difficulties inherent with a protein therapeutic approach to treating nervous system disorders, especially those of the CNS, increasing attention has now turned to the development of alternative strategies and, in particular, small molecule mimetics. Regulation of the transcription of neurotrophic factors may provide a means of manipulating endogenous factor production; gene therapy may also allow for the circumvention of exogenous neurotrophic factor administration. The problem of transport across the blood-brain barrier may be overcome by developing small-molecule mimetics that maintain the neurotrophic activity of the protein while having improved pharmacokinetic and disposition characteristics. Components of neurotrophic factor signal transduction pathways may provide additional targets for novel drugs that can induce or modulate the responses normally activated by the binding of the neurotrophic factor to its receptor. This review focusses on some of the major themes and lines of mechanistic and therapeutic advances in this fast-moving field of neuroscience.

Animals↗

PubMed1998

Structural mosaicism on the submicron scale in the plasma membrane.

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using single particle tracking (SPT). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode, or the size of their cytoplasmic domain, were expressed in National Institutes of Health 3T3 cells and C2C12 muscle cells. On a 6.6-s time scale, SPT measurements on both transmembrane and glycosylphosphatidylinositol (GPI) anchored isoforms of NCAM expressed in 3T3 cells could be classified into mobile (Brownian diffusion), slow diffusion, corralled diffusion, and immobile subpopulations. On a 90-s time scale, SPT studies in C2C12 cells revealed that 40-60% of transfected NCAM was mobile, whereas a smaller fraction (approximately 10-30%) experienced much slower diffusion. In addition, a fraction of approximately 30% of both transfected GPI and transmembrane isoforms and endogenous NCAM isoforms in C2C12 cells experienced transient confinement for approximately 8 s within regions of approximately 300-nm diameter. Diffusion within both these and the slow diffusion regions was anomalous, consistent with movements through a dense field of obstacles, whereas diffusion outside these regions was normal. Thus the membrane appears as a mosaic containing regions that permit free diffusion as well as regions in which NCAM is transiently confined to small or more extended domains. These results, including a large, freely diffusing fraction, similar confinement of transmembrane and GPI isoforms, a significant slowly diffusing fraction, and relatively large interdomain distances, are at some variance with the membrane skeleton fence model (Kusumi and Sako, 1996). Possible revisions to the model that incorporate these data are discussed.

3T3 Cells↗

PubMed1998

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

Animals↗

PubMed1998

Induction of experimental autoimmune neuritis with peripheral myelin protein-22.

Two myelin proteins, P2 basic protein and P0 glycoprotein, can induce experimental autoimmune neuritis (EAN), a model of human inflammatory neuropathy. We investigated whether peripheral nerve myelin protein-22 (PMP22), the gene for which is duplicated in hereditary motor sensory neuropathy type la, can also induce EAN. PMP22 cDNA produced by the reverse transcriptase-polymerase chain reaction from rat sciatic nerve was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). Ten Lewis rats were immunized with purified PMP22 fusion protein (50-100 microg) and eight controls with the same amount of GST. Two additional animals were immunized with each of two peptides (250 microg) of the human PMP22 extracellular sequences. Animals were examined daily until 20 days following immunization, when they underwent neurophysiological examination. A serum sample was then taken, prior to perfusion with glutaraldehyde and removal of the sciatic nerves and cauda equina. PMP22-immunized animals developed antibodies to the fusion protein and five out of 10 developed limp tails. No changes were observed in controls immunized with GST or in animals immunized with peptide. The mean compound motor action potentials elicited in the foot muscles by stimulation of the sciatic nerve at the sciatic notch and of the tibial nerve at the ankle were significantly reduced in the PMP22-immunized group (P < 0.05). Spinal roots from the group of animals immunized with PMP22 showed sparse infiltration of mononuclear cells, oedema and demyelination. PMP22 now deserves consideration as an autoantigen in human acute inflammatory demyelinating polyradiculoneuropathy.

Action Potentials↗

PubMed1997

Cellular determinants of the lateral mobility of neural cell adhesion molecules.

The lateral mobility of the neural cell adhesion molecule (NCAM) was examined using fluorescence recovery after photobleaching (FRAP). Various isoforms of human NCAM, differing in their ectodomain, their membrane anchorage mode or in the size of their cytoplasmic domain, were expressed in NIH 3T3 cells and C2C12 muscle cells. When the various isoforms were compared in 3T3 cells, FRAP studies showed both GPI-anchored and transmembrane isoforms diffused rapidly and only small differences in either the diffusion coefficients (D) or the mobile fractions (mf) were measured, suggesting the importance of the ectodomain in regulating lateral diffusion. However, the mobility of all NCAM isoforms was greatly reduced in regions of cell-cell contact, presumably due to homophilic trans interactions between NCAMs on adjacent cells. NCAM isoforms transfected into C2C12 cells which express NCAM naturally usually displayed a significantly lower D compared to the same isoforms transfected into 3T3 cells. Thus, NCAM lateral mobility is modulated in regions where cells interact and by the structure of the host cell membrane.

3T3 Cells↗

PubMed1997

Selective inhibition of growth factor-stimulated mitogenesis by a cell-permeable Grb2-binding peptide.

The activation of the mitogen-activated protein kinase (MAPK) cascade by a variety of growth factors and other agents is central to a mitogenic response. In the case of polypeptide growth factors such as the epidermal growth factor (EGF) and platelet-derived growth factor (PDGF), the steps leading to activation of MAPK require the function of the adaptor protein Grb2 (growth factor receptor binding protein 2), which can bind either directly or indirectly via its Src homology 2 domain to activated receptor tyrosine kinases. A cell-permeable mimetic of the EGF receptor Grb2 binding site has been investigated for its ability to inhibit biological responses stimulated by a variety of growth factors. Pretreatment of cells with this peptide results in the accumulation of the peptide in cells and its association with Grb2. This is associated with a complete inhibition of the mitogenic response stimulated by EGF and PDGF. In contrast, the peptide has no effect on the mitogenic response stimulated by fibroblast growth factor. The peptide could also inhibit the phosphorylation of MAPK stimulated with EGF and PDGF in the absence of an effect on the fibroblast growth factor response. These data demonstrate that cell-permeable mimetics of Src homology 2 binding sites can selectively inhibit growth factor-stimulated mitogenesis, and also directly demonstrate specificity in the coupling of activated receptor tyrosine kinases to the MAPK cascade.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Genetic correction of dystrophin deficiency and skeletal muscle remodeling in adult MDX mouse via transplantation of retroviral producer cells.

Duchenne muscular dystrophy (DMD) is an X-linked, lethal disease caused by mutations of the dystrophin gene. No effective therapy is available, but dystrophin gene transfer to skeletal muscle has been proposed as a treatment for DMD. We have developed a strategy for efficient in vivo gene transfer of dystrophin cDNA into regenerating skeletal muscle. Retroviral producer cells, which release a vector carrying the therapeutically active dystrophin minigene, were mitotically inactivated and transplanted in adult nude/mdx mice. Transplantation of 3 x 10(6) producer cells in a single site of the tibialis anterior muscle resulted in the transduction of between 5.5 and 18% total muscle fibers. The same procedure proved also feasible in immunocompetent mdx mice under short-term pharmacological immunosuppression. Minidystrophin expression was stable for up to 6 mo and led to alpha-sarcoglycan reexpression. Muscle stem cells could be transduced in vivo using this procedure. Transduced dystrophic skeletal muscle showed evidence of active remodeling reminiscent of the genetic normalization process which takes place in female DMD carriers. Overall, these results demonstrate that retroviral-mediated dystrophin gene transfer via transplantation of producer cells is a valid approach towards the long-term goal of gene therapy of DMD.

Animals↗

PubMed1997

Soluble myelin-associated glycoprotein (MAG) found in vivo inhibits axonal regeneration.

Myelin-associated glycoprotein (MAG) is a potent inhibitor of axonal regeneration when used as a substrate for growth. However, to be characterized definitively as inhibitory rather than nonpermissive, MAG must also inhibit axonal regeneration when presented in solution. Here, we show that soluble dMAG (extracellular domain only), released in abundance from myelin and found in vivo and chimeric MAG-Fc, can potently inhibit axonal regeneration. For both dMAG and MAG-Fc, inhibition is dose-dependent. If myelin-conditioned medium is immunodepleted of dMAG, or if a MAG antibody is included with MAG-Fc, inhibition is completely neutralized. Together with MAG's ability to induce growth cone collapse, these results demonstrate that MAG is an inhibitory molecule and not merely nonpermissive. The results also suggest that MAG binds to a specific receptor and initiates a signal transduction cascade to effect inhibition. Importantly, these results indicate that soluble dMAG detected in vivo could contribute to the lack of regeneration in the mammalian CNS after injury.

Animals↗

PubMed1997

Structural features of collapsin required for biological activity and distribution of binding sites in the developing chick.

We have used Fc-chimeras of collapsin-1/Sema III to study the structure-function activity of this recently identified repulsive axonal guidance molecule and to map the distribution of its binding sites during chick development. Our results show that the biological activity of the collapsin-Fc in an in vitro collapse assay is independent of both the Ig-domain and the positive charged carboxy terminus. Collapsin binding sites were found on a number of neuronal fiber tracts, and in two instances (DRG tracts and the retinotectal projection) this expression is both highly dynamic and consistent with them playing a role in axonal growth and guidance. Collapsin-1 binding sites were also found on a number of nonneuronal structures that do not produce collapsin-1 mRNA. We postulate that these sites may act to localize or concentrate collapsin-1 released from growing axons and in this way allow for an autocrine axonal guidance mechanism to function during development.

Animals↗

PubMed1997

Expression of a dominant negative FGF receptor inhibits axonal growth and FGF receptor phosphorylation stimulated by CAMs.

The cell adhesion molecules (CAMs) NCAM, N-cadherin, and L1 are homophilic binding molecules that stimulate axonal growth. We have postulated that the above CAMs can stimulate this response by activating the fibroblast growth factor receptor (FGFR) in neurons. In the present study, we demonstrate that activation of NCAM and L1 can lead to phosphorylation of the FGFR. Both this and the neurite outgrowth response stimulated by all three of the above CAMs are lost when a kinase-deleted, dominant negative form of FGFR1 is expressed in PC12 cells. In addition, we have generated transgenic mice that express the dominant negative FGFR under control of the neuron-specific enolase (NSE) promoter. We show that cerebellar neurons isolated from these mice have also lost their ability to respond to NCAM, N-cadherin, and L1. A peptide inhibitor of phospholipase C gamma (PLCgamma) that inhibits neurite outgrowth stimulated by FGF also inhibited neurite outgrowth stimulated by the CAMs. Thus, we conclude that activation of the FGFR is both necessary and sufficient to account for the ability of the above CAMs to stimulate axonal growth, and that PLCgamma is a key downstream effector of this response.

3T3 Cells↗

PubMed1997

Effect of NCAM-transfection on growth and invasion of a human cancer cell line.

A cDNA encoding the human transmembrane 140 kDa isoform of the neural cell adhesion molecule (NCAM) was transfected into the highly invasive MDA-MB-231 human breast cancer cell line. Transfectants with a homogeneous expression of NCAM showed a restricted capacity for penetration of an artificial basement membrane. However, when injected into nude mice, both control and NCAM-expressing cell lines produced equally invasive tumors. Tumors generated from NCAM-transfected cells were heterogeneous, containing NCAM-positive as well as NCAM-negative areas, indicating the existence of host factors capable of modulating NCAM expression in vivo. In nude mice, NCAM-transfected cells developed tumors with longer latency periods and slower growth rates than tumors induced by NCAM-negative control cells, implying that NCAM may be involved not only in adhesive and motile behavior of tumor cells but also in their growth regulation. There was no indication of differences in cell proliferative characteristics between the different NCAM-transfected and the control transfected cells as determined by flow cytometric DNA analysis, suggesting an increased cell loss as the reason for decreased in vivo growth rate of the NCAM-transfected cells. The fact that NCAM expression influences growth regulation attributes a pivotal role to this cell adhesion molecule during ontogenesis and tumor development.

Cell Cycle↗

PubMed1997

Neural cell adhesion molecules of the immunoglobulin superfamily: role in axon growth and guidance.

NCAM, L1, and DCC--immunoglobulin cell adhesion molecules (Ig CAMs)--are widely expressed during development. Many workers have dismissed a role for such molecules in the control of axonal growth and guidance because they do not show highly restricted expression patterns. Yet evidence from a number of model systems suggests all three CAMs play a role in the development of specific projections in the nervous system. For example, there is a reduction in mossy fiber tracts in the hippocampus of mice that lack NCAM, a requirement for DCC in the response of commissural neurons to a floor plate-derived chemoattractant, and a loss of corticospinal tracts in humans who carry mutations in the L1 gene. The above paradox might be explained by the observation that differential post-translational processing can modulate CAMs function and that alternative splicing can generate functionally distinct isoforms of a CAM. Activation of the FGF tyrosine kinase receptor is required for the responses stimulated by NCAM and L1, and the importance of regulated tyrosine phosphorylation for growth and guidance is underscored by the involvement of receptor tyrosine phosphatases in this process.

Animals↗