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M Schachner

Publications and source records attributed to M Schachner.

At least 127 records · Page 7Linked to original sources
PubMed1997

Expression cloning of a cDNA encoding a sulfotransferase involved in the biosynthesis of the HNK-1 carbohydrate epitope.

The HNK-1 carbohydrate epitope is expressed on several neural adhesion glycoproteins and as a glycolipid, and is involved in cell interactions. The structural element of the epitope common to glycoproteins and glycolipids has been determined to be sulfate-3-GlcAbeta1--> 3Galbeta1-->4GlcNAc. The glucuronyltransferase and sulfotransferase are considered to be the key enzymes in the biosynthesis of this epitope because the rest of the structure occurs often in glycoconjugates. Here we describe the isolation of the rat sulfotransferase cDNA via an expression cloning strategy. The clone finally isolated predicts a protein of 356 amino acids, with characteristics of a type II transmembrane protein and with no sequence similarity to other known sulfotransferases. Both the enzyme expressed as a soluble fusion protein and homogenates of cells transfected with the full-length cDNA could transfer sulfate from a sulfate donor to acceptor substrates containing terminal glucuronic acid.

Amino Acid Sequence↗
PubMed1997

Sodium channel distribution in axons of hypomyelinated and MAG null mutant mice.

Na+ channel organization was studied with immunofluorescence in the peripheral nervous system of mice genetically altered to produce abnormal myelin. In two of these strains, transcription of inserted transgenes was targeted to myelinating Schwann cells through linkage to a promoter for the myelin protein P0. Adults of both of these strains had hindlimb paralysis and a tremor on lifting by the tail. In one case, Schwann cells were eliminated via expression of the diphtheria toxin A chain (DT-A). During postnatal days 3-7, Na+ channel clustering at forming nodes was dramatically reduced compared with that of normal animals. At 1-3 months of age, Na+ channel immunofluorescence was often found spread over long stretches of the axolemma, instead of being confined to nodal gaps. In the second P0-linked transgenic model, Schwann cell expression of the large T antigen tsA-1609 resulted in cell cycle dysfunction. Adult axons had regions of diffuse Na+ channel labeling. Focal clusters were rare within these zones, which were characterized by a series of cells of myelinating phenotype tightly apposed to the axon. Previous studies suggested that Schwann cells had to reach the stage of ensheathment characterized by periaxonal myelin associated glycoprotein (MAG) expression in order to induce Na+ channel clustering. However, in MAG-deficient mice, Na+ channel labeling patterns within sciatic nerves were normal.

Aging↗
PubMed1997

VASE-encoded peptide modifies NCAM- and L1-mediated neurite outgrowth.

Interactions between the neural cell adhesion molecule (NCAM) with NCAM-expressing neurons (trans-interaction) stimulate outgrowth of neurites. The extent of NCAM-triggered neurite outgrowth depends on the presence of 10 amino acids derived from the variable alternatively spliced exon (VASE or pi-exon) in the fourth immunoglobulin-like domain of NCAM (Ig4): NCAM with VASE reduces and without VASE enhances neurite outgrowth in cis- or trans-interaction. We have investigated the role of VASE in neurite outgrowth by characterizing the receptors at the cell surface of cultured cerebellar neurons. Results from experiments with L1 and NCAM antibodies and with cerebellar neurons derived from wild-type or NCAM-deficient mice show that substrate-coated Ig4 with VASE (Ig4+) or without VASE (Ig4-) stimulates neurite outgrowth by a trans-interaction with L1 and that Ig4- promotes neurite outgrowth more strongly than Ig4+ by a transinteraction with NCAM.

Alternative Splicing↗
PubMed1997

The C-type lectin domains of lecticans, a family of aggregating chondroitin sulfate proteoglycans, bind tenascin-R by protein-protein interactions independent of carbohydrate moiety.

The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate-protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein-protein interaction through the fibronectin type III domains 3-5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein-protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein-protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.

Animals↗
PubMed1997

Signaling events following the interaction of the neuronal adhesion molecule F3 with the N-terminal domain of tenascin-R.

Interaction between the extracellular matrix protein tenascin-R and the neuronal adhesion molecule F3 might be involved in the formation of neuronal networks. In this study, the fragment of tenascin-R comprising epithelial growth factor (EGF)-like repeats and the cysteine-rich NH2 terminal stretch (EGF-L), known to be inhibitory for growing neurites and repellent for growth cones, was used to investigate the signaling events following the F3/EGF-L interaction. We addressed this question using an in vitro test with F3-transfected Chinese hamster ovary (CHO) cells that allowed us to measure the kinetics, magnitude and specificity of the repellent effect resulting from the specific F3/EGF-L interaction. We showed that the repellent effect was counteracted by addition of the serine/threonine kinase and -phosphatase modulators (staurosporine, okadaic acid and H7) but not by modulators of tyrosine kinase or -phosphatases. This result indicates that the intracellular signals activated by the repellent effect involve a serine/threonine kinase pathway. Furthermore, the repellent effect of the EGF-L fragment for growth cones of cultured cerebellar neurons was also abolished by the identical modulators of serine/threonine kinase and -phosphatases. The inhibition of neurite outgrowth from hippocampal neurons by EGF-L was abolished in the presence of the serine threonine-kinase inhibitor H7. These results strongly suggest that the F3/tenascin-R interaction through EGF-L involves an intracellular activation of serine/ threonine kinase(s) in all F3-expressing cells tested.

Animals↗
PubMed1997

Inhibitors of protein kinases abolish ECM-mediated promotion of neuronal polarity.

Different extracellular matrix (ECM) molecules, when presented to hippocampal neurons in culture in a substrate-bound form, exert strikingly similar effects on the establishment of neuronal polarity, i.e., the growth of axon-like major neurites is favored, whereas extension of dendrite-like minor neurites is inhibited. To gain insight into the underlying signal transduction processes, we have investigated the effects of modulators of protein kinase activity on the morphology of neurons cultured on tenascin-R, tenascin-C, and laminin-entactin substrates. We found differential effects of broad-spectrum protein kinase inhibitors: H-7 promoted the growth of minor neurites, whereas H-8 reduced the growth of major neurites on ECM but not control substrates. In contrast, chelerythrine, a specific inhibitor of protein kinase C, selectively affected growth of both minor and major neurites on control, but not on ECM substrates. Finally, reagents which elevate intracellular cAMP levels facilitated growth of minor neurites and inhibited growth of major neurites and thus interfered with the establishment of a polarized phenotype on both ECM and control substrates. Our results suggest that protein kinases mediate the effects of ECM molecules on neuronal polarity and that different kinases control extension of axons and dendrites.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Tenascin-C expression and axonal sprouting following injury to the spinal dorsal columns in the adult rat.

We have examined the expression and distribution of the extracellular matrix molecule tenascin-C in and around lesions of the thoracic dorsal columns in adult rats 3 days to 8 weeks after injury, using in situ hybridization, immunofluorescence, electron microscopy and immunoelectron microscopy. Numerous tenascin-C mRNA+ cells were present in and around the lesion at 3 days; fewer were present at 14 days and almost none 30 days after injury. Most tenascin-C mRNA+ cells in the spinal cord around the lesion were GFAP+, but most of those within the lesion were not, suggesting that tenascin-C is produced in the injured spinal cord by a subpopulation of astrocytes and by other cells that invade the lesion; these cells may include meningeal cells, macrophages, and Schwann cells. From 3 to 30 days after injury, heavy tenascin-C immunoreactivity was present at the lesion site (especially transections), and there was lighter immunoreactivity around the lesion and in the degenerating dorsal column. The heaviest immunoreactivity was associated with collagen fibrils in areas of expanded extracellular space and with basal laminae (covering Schwann cells and some astrocytes) but tenascin-C was also found close to the surfaces of some OX-42 + macrophages/microglia, leptomeningeal cells, and capillaries. Neurofilament (NF)+ axons grew into the highly tenascin-C-immunoreactive lesion sites, indicating that tenascin-C does not prevent axonal growth into these areas. However, such axons were not coated with tenascin-C except where directly exposed to the extracellular space.

Age Factors↗
PubMed1997

NCAM 180 in the postnatal development of cat visual cortex: an immunohistochemical study.

The neural cell adhesion molecule (NCAM) consists of three major isoforms with differing extents of intracellular domains. The largest, NCAM 180, may play an important role in the development and stabilization of cell contacts because of its interaction with the cytoskeleton, its accumulation at contact sites between cells, and age-dependent modifications of the immunoreactivity of its intracellular domain. The developmental expression pattern of NCAM 180 was examined in the cat visual cortex to explore its relation with the age-dependent decline of synaptic malleability. Western blot and immunohistochemical analyses were carried out by using a monoclonal antibody (D3) directed against an epitope of the intracellular domain specific to NCAM 180 and antibodies against all NCAM isoforms. The latter revealed a similar time course of increased expression during development by both Western blot and immunohistochemical analysis. In contrast, the D3 antibody showed an age-dependent increase of immunoreactivity by Western blot analysis and a decrease of reactivity by immunohistochemistry. In addition, the D3 antibody revealed characteristic developmental changes of immunoreactivity in the neuropil and distribution of immunopositive neuronal cell bodies in the different cortical laminae. The observations from this and another study (Kramer et al., 1997) suggest that the D3 antibody-specific NCAM 180 epitope is masked during the stabilization of connectivity.

Animals↗
PubMed1997

Developmentally regulated masking of an intracellular epitope of the 180 kDa isoform of the neural cell adhesion molecule NCAM.

The neural cell adhesion molecule NCAM is a cell surface glycoprotein that occurs in several isoforms. It was previously shown that the largest 180-kDa isoform of NCAM (NCAM 180) accumulates at sites of cell contact and in postsynaptic densities and may be responsible for the stabilization of cell contacts by its interaction with the membrane-cytoskeleton linker protein brain spectrin. In immunohistochemical studies on the expression of the NCAM 180, we noticed that two NCAM 180 specific monoclonal antibodies, termed 481 and D3, showed different patterns of immunoreactivity in sections of fresh-frozen adult mouse brain. Here we show that the D3-specific, but not the 481-specific epitope becomes inaccessible to the antibody during development of the hippocampal formation, coincident with the establishment of stable cell-cell contacts. In contrast, in the olfactory bulb with its continually regenerating olfactory nerve fibers, both NCAM 180 antibodies remain fully immunoreactive throughout development and adulthood. We also show that the D3-specific epitope becomes inaccessible in primary cerebellar neuron cultures with time in culture. Electrophoretic separation of hippocampal membrane proteins under nondenaturating conditions showed NCAM to be present in protein complexes of different molecular weights at different developmental stages. We propose that NCAM is involved in the formation of developmentally regulated, noncovalent complexes with as yet unknown partner molecules that could be responsible for the masking of the D3-specific epitope.

Animals↗
PubMed1997

Increased number of unmyelinated axons in optic nerves of adult mice deficient in the myelin-associated glycoprotein (MAG).

We have recently demonstrated that the formation of myelin sheaths in the optic nerve of young postnatal mice deficient in the myelin-associated glycoprotein (MAG) is retarded when compared to age-matched wild-type mice. In the present study, we analyzed whether impaired myelination of retinal ganglion cell axons is detectable in adult MAG mutants. In optic nerves of 2- and 9-month-old MAG-deficient mice, we observed a significantly increased number of unmyelinated axons compared to age-matched wild-type mice. At both ages, unmyelinated axons in optic nerves of MAG mutants were of small caliber. The number of unmyelinated axons decreased significantly in 9-month-old MAG mutants when compared to 2-month-old MAG mutants, indicative of a slow and long-lasting myelination of axons in the mutant. Our observations support the view that MAG is involved in the initiation of myelination in the CNS.

Animals↗
PubMed1997

Structural abnormalities and deficient maintenance of peripheral nerve myelin in mice lacking the gap junction protein connexin 32.

Mutations affecting the connexin 32 (Cx32) gene are associated with the X-linked form of the hereditary peripheral neuropathy Charcot-Marie-Tooth disease (CMTX). We show that Cx32-deficient mice develop a late-onset progressive peripheral neuropathy with abnormalities comparable to those associated with CMTX, thus providing proof of the critical role of Cx32 in the maintenance of peripheral nerve myelin and an animal model for CMTX. Frequently observed features include abnormally thin myelin sheaths, cellular onion bulb formation reflecting myelin degeneration-induced Schwann cell proliferation, and enlarged periaxonal collars while nerve conductance properties are altered only slightly. These observations are consistent with earlier hypotheses suggesting a function of Cx32 as a channel-forming protein that facilitates the communication between the abaxonal and adaxonal aspects of Schwann cell cytoplasm.

Afferent Pathways↗
PubMed1997

Tenascin-R is an intrinsic autocrine factor for oligodendrocyte differentiation and promotes cell adhesion by a sulfatide-mediated mechanism.

O4(+) oligodendrocyte (OL) progenitors in the mammalian CNS are committed fully to terminal differentiation into myelin-forming cells. In the absence of other cell types in vitro, OL differentiation reproduces the in vivo development with a correct timing, suggesting the existence of an intrinsic regulatory mechanism that presently is unknown. We have examined the effect of two isoforms of the extracellular matrix (ECM) molecule tenascin-R (TN-R), which is expressed by OLs during the process of myelination, on the adhesion and maturation of OLs in vitro. Here we show that the substrate-bound molecules supported the adhesion of O4(+) OLs independently of the CNS region or age from which they were derived. At the molecular level this process was mediated by protein binding to membrane surface sulfatides (Sulf), as indicated by the interference of O4 antibody and Sulf with the attachment of OLs or other Sulf+ cells, erythrocytes, to TN-R substrates and by direct protein-glycolipid binding studies. In the absence of platelet-derived growth factor (PDGF), exogenous TN-R induced myelin gene expression and the upregulation of its own synthesis by cultured cells, resulting in a rapid terminal differentiation of O4(+) progenitors. Our findings strongly suggest that TN-R represents an intrinsic regulatory molecule that controls the timed OL differentiation by an autocrine mechanism and imply the relevance of TN-R for CNS myelination and remyelination.

Animals↗
PubMed1997

HNK-1 carbohydrate-mediated cell adhesion to laminin-1 is different from heparin-mediated and sulfatide-mediated cell adhesion.

The sulfated HNK-1 carbohydrate present on glycolipids and on several neural recognition molecules has been shown to mediate the adhesion of murine small cerebellar neurons and astrocytes to the extracellular matrix molecule laminin-1. In this study, we characterized the binding of the HNK-1 carbohydrate to laminin-1 extracted from the Engelbreth-Holm-Swarm (EHS) sarcoma and distinguished it unequivocally from binding sites for other sulfated carbohydrates. Electron microscopic analysis of rotary shadowed complexes of laminin-1 and a HNK-1 neoglycoprotein revealed a major binding site on the G domain that comprises the C-terminal globule of the laminin alpha1 chain. The HNK-1 carbohydrate also interacted with placental laminin isoforms containing an alpha chain variant. It bound to the proteolytic laminin-1 fragment E8 comprising the domains G1-G3, but not to fragment E3 that carries the major heparin-binding site on domains G4-G5. No binding was observed to the short arm containing fragments E1XNd or P1. Binding studies with native or denatured laminin E8 fragments and proteolytic or recombinant fragments of the G domain localized the HNK-1 carbohydrate binding site to domain G2. The binding could be clearly distinguished from binding sites for other sulfated carbohydrates such as heparin and sulfatides. Further, the binding could not be abolished by reduction and alkylation or by urea treatment of laminin-1 and was independent of the native conformation of laminin-1 and of Ca2+. The G2 domain is also involved in the adhesion of HNK-1 carbohydrate expressing early postnatal cerebellar neurons and is different from heparin- and sulfatide-mediated cell adhesion to laminin-1. HNK-1 carbohydrate-mediated cell adhesion appears, however, to be dependent on the native conformation of laminin-1 indicating a more complex cellular recognition process.

Animals↗
PubMed1997

Tyrosine phosphorylation of L1 family adhesion molecules: implication of the Eph kinase Cek5.

The L1 family comprises transmembrane cell adhesion molecules of the immunoglobulin superfamily that play an important role in neuronal migration and axon outgrowth, fasciculation, and myelination. Consistent with a crucial role in developmental processes, mutations in L1 cause severe brain malformations. Although L1 activates intracellular signaling pathways, little is known about the membrane proximal events of L1 signaling. The cytoplasmic domains of L1 family proteins contain several conserved tyrosine residues that are potential targets for receptor tyrosine kinases. Here, we report that the L1 family protein Ng-CAM is phosphorylated on tyrosine in embryonic day 13 chicken retina. This is the first demonstration of in vivo tyrosine phosphorylation of an L1-like molecule. Because chicken embryo kinase 5 (Cek5) is a receptor tyrosine kinase expressed in neuronal processes and activated in the chicken embryonic retina, we have analyzed the possible role of Cek5 in L1 phosphorylation. The rat glioblastoma cell line B28 was stably transfected with human L1. Additional transient transfection with Cek5 cDNA led to expression of Cek5 in its tyrosine-phosphorylated, activated form. Biochemical analysis revealed that L1 is phosphorylated on tyrosine in Cek5-transfected cells but not in control transfectants. Furthermore, direct phosphorylation of the L1 cytoplasmic domain by Cek5 was demonstrated in an in vitro kinase assay. Tyrosine phosphorylation may represent a novel mechanism of signal cascade initiation through L1.

Amino Acid Sequence↗
PubMed1997

Axonal regrowth after spinal cord transection in adult zebrafish.

Using axonal tracers, we characterized the neurons projecting from the brain to the spinal cord as well as the terminal fields of ascending spinal projections in the brain of adult zebrafish with unlesioned or transected spinal cords. Twenty distinct brain nuclei were found to project to the spinal cord. These nuclei were similar to those found in the closely related goldfish, except that additionally the parvocellular preoptic nucleus, the medial octavolateralis nucleus, and the nucleus tangentialis, but not the facial lobe, projected to the spinal cord in zebrafish. Terminal fields of axons, visualized by anterograde tracing, were seen in the telencephalon, the diencephalon, the torus semicircularis, the optic tectum, the eminentia granularis, and throughout the ventral brainstem in unlesioned animals. Following spinal cord transection at a level approximately 3.5 mm caudal to the brainstem/spinal cord transition zone, neurons in most brain nuclei grew axons beyond the transection site into the distal spinal cord to the level of retrograde tracer application within 6 weeks. However, the individually identifiable Mauthner cells were never seen to do so up to 15 weeks after spinal cord transection. Nearly all neurons survived axotomy, and the vast majority of axons that had grown beyond the transection site belonged to previously axotomized neurons as shown by double tracing. Terminal fields were not re-established in the torus semicircularis and the eminentia granularis following spinal cord transection.

Afferent Pathways↗
PubMed1997

Advances in Charcot-Marie-Tooth disease research: cellular function of CMT-related proteins, transgenic animal models, and pathomechanisms. The European CMT Consortium.

The First Workshop of the European Consortium on Charcot-Marie-Tooth (CMT) disease brought together neuroscientists, molecular and cell biologists, neuropathologists, neurologists, and geneticists with a common interest in the understanding of the fundamental mechanisms that underlie the pathogenesis of CMT. The interdisciplinary group of 25 expert scientists discussed recent advances in (i) molecular genetics and histopathology of CMT, (ii) development of suitable animal models, (iii) understanding of the cellular function of CMT-related proteins, and (iv) studies using nerve biopsies from CMT patients. In this minireview, we summarize the key findings presented and discuss their impact on CMT research.

Animals↗
PubMed1997

Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance, but not with the formation of peripheral myelin.

We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG-/N-CAM- mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG-/N-CAM- than in MAG- mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG-/N-CAM- mutants when compared to MAG- mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity.

Animals↗