Biomedical subjects

M Schachner

Publications and source records attributed to M Schachner.

At least 181 records · Page 10Linked to original sources

PubMed1995

Immunohistological localization of tenascin-C in the developing and regenerating retinotectal system of two amphibian species.

The expression pattern of the extracellular matrix molecule tenascin-C was investigated in the retinotectal system of the frog Discoglossus pictus and the salamander Pleurodeles waltl during development and optic nerve regeneration in the adult. In both species, the retina was devoid of tenascin-C immunoreactivity at all ages studied. During development, tenascin-C was distributed in a gradient in the optic nerve, with the highest immunoreactivity in the eye near part of the optic nerve. The myelin-associated glycoprotein was distributed in a gradient with opposite polarity. In Discoglossus, but not Pleurodeles, tenascin-C was detected in the anterior chiasm. In the tectum of both species, tenascin-C was observed in deep cellular and fiber layers but not in the layers receiving optic fibers or proliferative zones. The distribution patterns of tenascin-C were the same during development and in the adult, except for a disappearance of the molecule from the intraocular part of the optic nerve. After lesioning the optic nerve of adult animals, tenascin-C was strongly reexpressed in the intraocular part of the optic nerve but was only weakly upregulated in the distal optic nerve stump. In contrast, a chondroitin sulfate epitope was strongly upregulated in the distal optic nerve stump. These observations suggest that during development, tenascin-C serves as an attenuating barrier for myelinating cells in the optic nerve and contributes to the guidance of growing retinal ganglion cell axons. Due to its sustained expression in the adult, tenascin-C may have similar functions during regeneration of the lesioned adult retinotectal system.

Animals↗

PubMed1995

Control of neuronal morphology in vitro: interplay between adhesive substrate forces and molecular instruction.

Among the factors which influence neuronal morphology, the degree of substrate adhesivity has been suggested to play an important role in the growth and guidance of neurites. The present study was undertaken to investigate apparently contradictory results relating substrate adhesivity to the extent of neurite outgrowth. By using substrates coated with different concentrations of polyornithine to vary adhesivity, we could show that intermediate levels of neuron-to-substrate adhesive strength favored neurite outgrowth more than substrates of high or low adhesivity. However, when neurons were plated on substrates derived from the extracellular matrix, the strength of neuron-to-substrate adhesion was important for the growth of dendrite-like minor neurites, but not for the extension of axon-like major neurites, which grew independently of adhesive forces. On substrates of the cell adhesion molecule L1, growth of both major and minor neurites was adhesion-independent. Finally, in the presence of tenascin added to the culture medium, neurite growth was inhibited irrespective of the adhesivity of the substrate and the presence of substrate-bound extracellular matrix molecules or L1. These observations suggest that intermediate forces of adhesivity favor neurite growth in general, but that purely adhesive forces can be dominated by specific molecular instructions which differentially affect growth of major and minor neurites in positive and negative ways.

Animals↗

PubMed1995

Adhesive hierarchy involving the cell adhesion molecules L1, CD24, and alpha 6 integrin in murine neuroblastoma N2A cells.

The aggregation rate of resuspended neuroblastoma N2A cells depends on the density of the cells in culture prior to their resuspension: isolated, fast growing cells have a weak tendency to aggregate whereas confluent, slowly growing cells reaggregate very strongly. L1 antibody 557 strongly inhibited the slow aggregation of isolated, fast growing cells but not the reaggregation of confluent cells. CD24 (nectadrin) antibodies did not affect the aggregation of isolated or confluent cells but stimulated the aggregation of subconfluent cells. In all stages aggregation was not inhibited when antibody 557 was used together with CD24 antibodies at 37 degrees C in the presence of divalent cations. EA-1 antibody to alpha 6 integrin chain stimulated the aggregation of subconfluent cells but inhibited the reaggregation of confluent cells. Therefore, L1 appears to be an early recognition molecule mediating weak primary adhesion. CD24 appears to participate in activating secondary adhesion mechanisms during primary adhesion, possibly in cooperation with L1, and alpha 6 integrin seems to serve as a secondary, strong adhesion molecule that in early adhesion phases also mediates the activation of itself or of other adhesion mechanisms. These results indicate that neural cells might employ a strategy of adhesion cascade in establishing stable contacts.

Animals↗

PubMed1995

Several extracellular domains of the neural cell adhesion molecule L1 are involved in homophilic interactions.

The neural cell adhesion molecule L1 is a multidomain protein that plays important roles in cell adhesion, migration, and neurite outgrowth. It can interact with itself by a self-binding, i.e., homophilic adhesion mechanism (Kadmon et al.: J Cell Biol 110: 193-208, 1990a). To determine the domains of L1 involved in homophilic binding, we have generated protein fragments of L1 in a prokaryotic and a eukaryotic expression system and used these covalently coupled to fluorescent microspheres to quantify aggregation between them by cytofluorometric analysis. Protein fragments containing the first and second Ig-like domains and the third fibronectin type III homologous repeat showed avid self-binding. Ig-like domains III and IV also showed some self-binding, whereas Ig-like domains V and VI and fibronectin type III homologous repeats 1 and 2 as well as 4 and 5 were less or not active. Binding between different domains was also observed: fibronectin type III homologous repeats 4 and 5 interacted with Ig-like domains I and II, and fibronectin type III homologous repeats 3-5 interacted with all Ig-like domains. These results were confirmed by experiments testing the binding of fragment-conjugated microspheres to substrate-coated L1 or to cell surface-expressed L1 on cultured neurons. Binding of L1 to itself was interfered with by all protein fragments tested, suggesting that also less avidly binding domains of L1 contribute to homophilic binding. These observations indicate prominent functional roles of both Ig-like domains and fibronectin type III homologous repeats in homophilic binding of L1.

Animals↗

PubMed1995

Behaviour of small inhibitory interneurons in early postnatal mouse cerebellar microexplant cultures: a video time-lapse analysis.

The aim of this work was to investigate how the environment of the neuropil determines the positioning and differentiation of neurons that are postsynaptic to them. We investigated how stellate and basket cells, the small inhibitory interneurons of the cerebellar cortex, find their perpendicular orientation to the direction of fasciculated granule cell axons. Cultures of early postnatal mouse cerebellar microexplants showing this cellular behaviour in vitro were analysed by video time-lapse cinematography and evaluated by morphometry. The small interneurons were first detectable when they migrated, intermingled with granule cells, away from the explant along the radial fascicles of granule cell neurites. During migration some cells suddenly changed their orientation by extending neurites in perpendicular orientation to the radial fascicles. These cells were all GABA-immunoreactive and expressed the cytoskeletal markers tau in the thin axon-like process and MAP2 in the thicker dendrite-like arborizations at the opposite pole of the cell body. After having translocated in perpendicular orientation, these neurons were again able to turn back to move along the radial neurite bundles to another position. Furthermore, while in perpendicular orientation, the processes of these cells repelled each other upon contact of their growth cones, leading to equal spacing between the cell bodies with time in culture.

Animals↗

PubMed1995

Fasciculation of granule cell neurites is responsible for the perpendicular orientation of small inhibitory interneurons in mouse cerebellar microexplant cultures in vitro.

To study the cellular and molecular mechanisms involved in the perpendicular orientation of stellate and/or basket cells to the direction of fasciculating granule cell neurites, we have used cultures of microexplants from early postnatal mouse cerebellum that show this cellular behaviour in vitro. When these cultures were maintained in the presence of antibodies to the neural cell adhesion molecules L1 and N-CAM or immunoglobulin-like domains I, II and IV of N-CAM, the resulting decrease or increase in the fasciculation of granule cell neurites changed the perpendicular orientation and morphology of the small inhibitory interneurons. Additives which did not perturb fasciculation did not affect the perpendicular orientation and morphology of stellate and/or basket cells. Furthermore, when perturbation of fasciculation was prevented, neither L1 nor N-CAM antibodies modified the positioning or morphology of interneurons. These observations indicate that ordered fasciculation of granule cell neurites is an important parameter in the perpendicular orientation and elaboration of the typical morphology of the small cerebellar inhibitory interneurons.

Animals↗

PubMed1995

Functional topography of myelin-associated glycoprotein. II. Mapping of domains on molecular fragments.

The myelin-associated glycoprotein (MAG), an adhesion molecule of the immunoglobulin (Ig) superfamily with five Ig-like domains, was investigated with regard to its binding site(s) for the neuronal cell surface, collagen I, and heparin, using a panel of new monoclonal antibodies and cyanogen bromide cleavage fragments of MAG. All antibodies generated competed with each other for binding to MAG, indicating that they reacted with identical or closely related epitopes. Mapping of the reactive epitopes on recombinant deletion fragments of MAG expressed by Chinese hamster ovary (CHO) fibroblasts showed reactivity of monoclonal antibody 513 with domains I, II, and III, comprising the amino-terminal end of the extracellular domain. Monoclonal antibody 15 recognized domain III only. Binding of MAG-containing liposomes to neurons was blocked by antibodies 15 and 513. Cyanogen bromide (CNBr) fragments of domains I, II, and III bound to collagen type I under isotonic buffer conditions. CNBr fragments containing domains I and II were involved in binding to heparin. These observations suggest that domain III may be important for binding to the neuronal cell surface receptor for MAG, while domains I, II, and III interact with collagen type I and domains II and III with heparin.

3T3 Cells↗

PubMed1995

The neurotrophin receptors TrkA and TrkB are inhibitory for neurite outgrowth.

To investigate the possibility that the neurotrophin tyrosine kinase receptors are also recognition molecules by virtue of their immunoglobulin-like domains, the ability of TrkA and TrkB to influence neurite outgrowth was tested in vitro. Cell monolayers of fibroblasts transfected to express either the TrkA or TrkB receptor reduced neurite outgrowth of phaeochromocytoma PC12 cells by 50-60% when compared to mock transfected fibroblasts or fibroblasts transfected with the epidermal growth factor receptor. Neurite outgrowth from cerebellar neurons was inhibited by 30-40% on these substrates. When a recombinantly expressed fragment of TrkA comprising the two immunoglobulin-like domains was coated as a substrate in combination with poly-L-lysine and laminin, neurite outgrowth was inhibited in a dose-dependent manner. This inhibition of neurite outgrowth was not mediated via an interaction with laminin as there is no specific binding of the TrkA fragment to laminin. The adhesion of cell bodies to this substrate was not affected by the immunoglobulin-like domains. These observations suggest that the mammalian neurotrophin receptors not only influence neurite outgrowth by neurotrophin triggered activation of the receptor, but also by cell surface recognition processes conveyed by the immunoglobulin-like domains.

Animals↗

PubMed1995

The extracellular matrix molecule tenascin: expression in the developing chick retinotectal system and substrate properties for retinal ganglion cell neurites in vitro.

To investigate the molecular mechanisms involved in the outgrowth of retinal ganglion cell axons in the tectum, the expression of the extracellular matrix molecule tenascin was analysed in the tectum and retina of chickens by immunocytochemistry and in situ hybridization. Tissue was analysed between embryonic days 4 and 12, just before and during the period when retinal ganglion cell axons innervate their target region, the optic tectum. In the tectum, tenascin immunoreactivity becomes detectable at the anterior pole at embryonic day 4, 2 days before retinal ganglion cell axons arrive, and spreads caudally with increasing age. At early stages, tenascin is predominantly accumulated in the stratum opticum, the zone of ingrowing retinal ganglion cell axons, and along their prospective pathway. In the stratum opticum, the molecule is associated with radial glial fibres, glial endfeet and retinal ganglion cell axons located in the immediate neighbourhood of radial glial fibres. At all ages investigated, tenascin mRNA is mainly restricted to cells located in the periventricular region, suggesting that the molecule is synthesized by radial glial cells. In the retina, tenascin is expressed by amacrine, displaced amacrine and horizontal cells but not by retinal ganglion cells. To investigate whether the accumulation of tenascin in the developing and prospective pathway of retinal ganglion cell axons may affect their rate of growth we assayed the substrate properties of tenascin for retinal ganglion cell neurites in vitro. When retinal ganglion cell suspensions from 6-day-old chick embryos were maintained on homogeneous mouse or chick tenascin/polyornithine substrates, neurite length was significantly increased when compared to polyornithine substrates at coating concentrations of 10 or 20 micrograms/ml. Higher coating concentrations (35 or 70 micrograms/ml) resulted in neurite lengths comparable to control values. Together, these observations suggest that tenascin in the developing and prospective stratum opticum might serve as a performed pathway to support growth of retinal ganglion cell axons in the tectum.

Animals↗

PubMed1995

Evidence for cis interaction and cooperative signalling by the heat-stable antigen nectadrin (murine CD24) and the cell adhesion molecule L1 in neurons.

L1 is a transmembranal homophilic cell adhesion molecule of the immunoglobulin superfamily expressed by neural and lymphoid cells. The heat-stable antigen (HSA, murine CD24) nectadrin is a highly and heterogeneously glycosylated glycophosphatidylinositol-linked differentiation antigen of haematopoietic and neural cells. L1 and nectadrin have been shown to mediate cell adhesion and intracellular Ca2+ signals in neurons and B lymphoblasts, respectively. Here we show that nectadrin is co-expressed with L1 in murine cerebellar granule cell neurons and neuroblastoma N2A cells. Purified nectadrin bound to L1 with an apparent binding ratio of five nectadrin molecules to one L1 molecule at saturation. Binding between nectadrin and purified N-CAM was not observed. In co-capping experiments nectadrin co-redistributed with L1 and N-CAM. Since in these cells N-CAM and L1 cohere by cis-binding nectadrin appears to join the L1-N-CAM complex through binding to L1. Antibodies to each L1 and nectadrin evoked small increases in the intracellular Ca2+ concentration. However, when both antibodies were added together or in tandem to the cells, a strong intracellular Ca2+ signal was measured that was at least 6- and 10-fold stronger than the signal separately induced by L1 and nectadrin antibodies respectively. Such a cooperative effect was not observed in B lymphoblasts, using the same antibodies, or in neurons, using a combination of L1 and Thy-1 antibodies. Both the weak Ca2+ signal mediated by L1 alone and the enhanced signal jointly triggered by antibodies to L1 and nectadrin were inhibited by phorbol 12-myristate 13-acetate and were not significantly affected by Ni2+ and Cd2+ cations, suggesting that they are related to one another and involve recruitment of intracellular Ca2+. Nectadrin therefore appears to join a functional complex of neuronal adhesion molecules and to potentiate the signal transduction pathway of L1, possibly in response to neuron-neuron contact formation.

Animals↗

PubMed1995

Up-regulation of a chondroitin sulphate epitope during regeneration of mouse sciatic nerve: evidence that the immunoreactive molecules are related to the chondroitin sulphate proteoglycans decorin and versican.

After transection of adult mouse sciatic nerve, the expression of a chondroitin sulphate epitope recognized by the monoclonal antibody 473-HD (mAb 473-HD) was found to be up-regulated. The epitope was localized immunocytochemically mainly in Schwann cell basal laminae and, more weakly, also in the endoneurium. In cultures of mouse dorsal root ganglion cells, Schwann cells expressed high levels but fibroblasts only low levels of the epitope. To identify the molecule(s) carrying this chondroitin sulphate epitope, human sciatic nerves were extracted with phosphate-buffered saline and shown to contain two chondroitin sulphate proteoglycans of apparent molecular weights of 130 and 900 kDa. The 900 kDa and, more weakly, the 130 kDa proteoglycan were reactive with mAb 473-HD, which was found to recognize chondroitin-6-sulphate as epitope. Following chondroitinase ABC treatment of the 130 kDa proteoglycan, a core protein of approximately 45 kDa was seen and shown to react with polyclonal antibodies against the chondroitin-dermatan sulphate proteoglycan decorin from human fibroblasts. Chondroitinase ABC treatment of the 900 kDa proteoglycan yielded a core protein with a molecular weight of approximately 400 kDa that was recognized by polyclonal antibodies against recombinantly expressed fusion proteins from human versican. After transection of adult mouse sciatic nerves, the distal nerve stumps showed up-regulation of the chondroitin-6-sulphate epitope of the 900 kDa proteoglycan, whereas the core protein of this proteoglycan did not show any detectable change in the level of expression. In contrast, the core protein of the 130 kDa proteoglycan was up-regulated in expression. These observations suggest that versican- and decorin-like molecules may contribute to successful regeneration in the peripheral nervous system of mammals.

Animals↗

PubMed1995

Functional involvement of sciatic nerve-derived versican- and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth.

We have previously described two proteoglycans from human sciatic nerve which are immunochemically related to the chondroitin sulphate proteoglycans versican and decorin. The chondroitin sulphate of the versican-like molecule and the core protein of the decorin-like molecule have been found previously to be up-regulated after lesioning the adult mouse sciatic nerve. To investigate if the versican- and decorin-like molecules are involved in cell-extracellular matrix interactions, we studied the effect of both molecules on cell adhesion. The versican- and decorin-like molecules, substrate-coated in a mixture with fibronectin, but not with laminin or collagen types I or IV, inhibited the adhesion of several cell lines, neonatal dorsal root ganglion neurons and Schwann cells. The inhibitory activity was concentration-dependent and mediated by the chondroitin sulphate. Furthermore, when different proteoglycans were incubated with fibronectin, only the versican- and decorin-like molecules and the chondroitin sulphate proteoglycan aggrecan, but not the heparan sulphate proteoglycan perlecan, were able to inhibit fibronectin-mediated cell adhesion. The versican- and decorin-like molecules, substrate-coated alone or in a mixture with fibronectin or laminin, were at most slightly inhibitory to neurite outgrowth from PC12 phaeochromocytoma cells and neonatal dorsal root ganglion neurons. In a solid-phase ligand-binding assay the versican- and decorin-like molecules interacted with fibronectin, but not with laminin or collagen types I and IV. Binding of the versican-like molecule to fibronectin and inhibition of cell adhesion by this molecule was mediated via the heparin and cell-binding domains of fibronectin. These observations suggest that binding of the two proteoglycans to fibronectin is involved in the modulation of adhesion of cells to fibronectin.

Animals↗

PubMed1995

Recognition molecules myelin-associated glycoprotein and tenascin-C inhibit integrin-mediated adhesion of neural cells to collagen.

Because of the importance of collagens in mediating cell-substrate interactions and the association of collagens with neural recognition molecules in the peripheral nervous system, the ability of neural recognition molecules to modify the substrate properties of collagens, in particular collagen type I, for cell adhesion was determined. Two cell lines, the N2A neuroblastoma and PC12 pheochromocytoma, were investigated for their capacity to adhere to different collagen types in the absence or presence of several neural recognition molecules. Adhesion of N2A or PC12 cells and membrane vesicles from PC12 cells to collagen type I was reduced when the collagen had been preincubated prior to its application as substrate with the extracellular domain of myelin-associated glycoprotein (s-MAG) or, as control, fibroblast tenascin-C (F-tenascin). In mixture with other collagen types, s-MAG was only able to reduce the adhesiveness of collagen types III and V, but not of collagen types II and IV. F-tenascin reduced the adhesiveness of all collagen types tested. In contrast to F-tenascin, s-MAG had to be present during fibrillogenesis to exert its effect, indicating that it must be coassembled into the collagen fibril to block the binding site. Cell adhesion to collagen type I was dependent on Mg2+ or Mn2+ and inhibited by a monoclonal antibody to the alpha 1 integrin subunit. The combined observations indicate that s-MAG and F-tenascin interfere with cell binding, most probably by modifying the integrin binding site, and that the two molecules act by different mechanisms, both leading to reduction of adhesion.

Animals↗

PubMed1995

Crucial role for the myelin-associated glycoprotein in the maintenance of axon-myelin integrity.

It has recently been shown that mice deficient in the gene for myelin-associated glycoprotein develop normal myelin sheaths in the peripheral nervous system. Here we report that in mutant mice older than 8 months the maintenance of axon-myelin units is disturbed, resulting in both axon and myelin degeneration. Morphological features include those typically seen in human peripheral neuropathies, where demyelination-induced Schwann cell proliferation and remyelination lead to the formation of so-called onion bulbs. Expression of tenascin-C, a molecule indicative of peripheral nerve degeneration, was up-regulated by axon-deprived Schwann cells and regenerating axons were occasionally seen. Myelin-associated glycoprotein thus appears to play a crucial role in the long-term maintenance of the integrity of both myelin and axons.

Animals↗

PubMed1995

Exogenous testosterone alters expression pattern of cell recognition molecules in brains of juvenile zebra finches.

We assessed spatio-temporal expression of tenascin and janusin by immunocytochemistry in testosterone-treated zebra finches and in untreated controls. These cell recognition molecules and components of the extracellular matrix mediate neurone-glia interactions, which are crucial for differentiation of ordered neural structures. We describe boundaries for the spatio-temporal expression of cell recognition molecules that highly correlate with the song motor centres as defined by combined neuroanatomical and ethological studies on song vocalization. The expression of janusin is accelerated by exogenous testosterone.

Age Factors↗

PubMed1995

Clustering and co-localization of immunogold double labelled neural cell adhesion molecule isoforms in chick forebrain.

A quantitative immunoelectron microscopic study was carried out in the intermediate portion of hyperstriatum ventrale of chick forebrain (a region exhibiting learning-related morphological plasticity) in order to examine the fine distribution of the neural cell adhesion molecule (N-CAM). Antibodies against alpha 2,8 polysialic acid (PSA), characteristic for embryonic N-CAM, and those against the protein backbones of all N-CAM isoforms were sequentially labelled (post-embedding) with 5- and 15-nm gold particles, and binding to their epitopes was analyzed using the statistics of point processes. The results showed that: (1) PSA tends to form clusters (up to 200 nm in size), in contrast to the protein backbones of all N-CAM isoforms, and (2) the two tissue bound antibodies are co-localized spatially in clusters of sizes up to 100 nm. The data suggest that there is a spatial sub-cellular domain enriched in both PSA and protein isoforms of N-CAM.

Animals↗

PubMed1995

Multiply myelinated axons in the optic nerve of mice deficient for the myelin-associated glycoprotein.

We recently reported that some retinal ganglion cell axons in mice deficient for the myelin-associated glycoprotein are concentrically surrounded by more than one myelin sheath. In the present study, we demonstrate that myelin sheaths displaced from the axon reveal a normal ultrastructure of compact myelin, with the only exception that multiple myelination of axons frequently correlates with the presence of unfused regions of major dense lines. Supernumerary sheaths terminated on other sheaths or on astrocyte cell surfaces in a pattern closely resembling the morphology of a true paranode. The thickness of compact myelin of multiply myelinated axons was significantly increased when compared with axons of similar caliber surrounded by a single myelin sheath. Our observations demonstrate that maintenance of compact myelin and paranodal regions is not dependent on direct axonal contact and that the presence of more than one concentric myelin sheath around an axon results in dysregulation of the axon-to-fiber ratio.

Animals↗

PubMed1995

The AMOG/beta 2 subunit of Na,K-ATPase is not necessary for long-term survival of telencephalic grafts.

Adhesion molecule on glia (AMOG) represents the beta 2-subunit of murine Na,K-ATPase. Mice carrying a targeted deletion of the AMOG/beta 2 gene exhibit tremor and limb paralysis at postnatal day (P) 15 and die 2 days after the onset of symptoms. The brains of these mice show edema and swelling of astrocytic end feet. However, the cause of death has remained unclear. To identify long-term consequences of AMOG/beta 2 deficiency, we have grafted parts of the embryonic telencephalic anlage of AMOG/beta 2-deficient mice into the caudoputamen of wild-type mice and analyzed the grafts up to 500 days after transplantation. Histological, immunocytochemical, and in situ hybridization techniques were applied to examine histoarchitecture, proliferation, differentiation, and long-term survival of grafts. AMOG/beta 2-deficient telencephalic grafts develop normally and form solid neural tissue that cannot be distinguished from control grafts by morphological features or with immunocytochemical stains for neuronal and glial markers. No signs of degeneration can be found. Expression analysis, however, revealed that no AMOG/beta 2 protein of possible host origin can be detected in AMOG/beta 2-deficient grafts. Graft-borne astrocytes express neither the AMOG/beta 1 nor the AMOG/beta 2 subunit of Na,K-ATPase as examined with immunocytochemistry and in situ hybridization. These findings indicate that AMOG/beta 2 is not necessary for long-term survival of telencephalic graft tissue.

Adenosine Triphosphatases↗