Biomedical subjects

M Schachner

Publications and source records attributed to M Schachner.

At least 397 records · Page 22Linked to original sources

PubMed1986

Expression of several adhesive macromolecules (N-CAM, L1, J1, NILE, uvomorulin, laminin, fibronectin, and a heparan sulfate proteoglycan) in embryonic, adult, and denervated adult skeletal muscle.

Levels of the neural cell adhesion molecule N-CAM in muscle are regulated in parallel with the susceptibility of muscle to innervation: N-CAM is abundant on the surface of early embryonic myotubes, declines in level as development proceeds, reappears when adult muscles are denervated or paralyzed, and is lost after reinnervation (Covault, J., and J. R. Sanes, 1985, Proc. Natl. Acad. Sci. USA, 82:4544-4548). Here we used immunocytochemical methods to compare this pattern of expression with those of several other molecules known to be involved in cellular adhesion. Laminin, fibronectin, and a basal lamina-associated heparan sulfate proteoglycan accumulate on embryonic myotubes after synapse formation, and their levels change little after denervation. L1, J1, nerve growth factor-inducible large external protein, uvomorulin, and a carbohydrate epitope (L2/HNK-1) shared by several adhesion molecules are undetectable on the surface of embryonic, perinatal, adult, or denervated adult muscle fibers. Thus, of the molecules tested, only N-CAM appears on the surface of muscle cells in parallel with the ability of the muscle cell surface to accept synapses. However, four antigens--N-CAM, J1, fibronectin, and a heparan sulfate proteoglycan--accumulate in interstitial spaces near denervated synaptic sites; regenerating axons traverse these spaces as they preferentially reinnervate original synaptic sites. Of particular interest is J1, antibodies to which block adhesion of central neurons to astrocytes (Kruse, J., G. Keihauer, A. Faissner, R. Timpl, and M. Schachner, 1985, Nature (Lond.), 316:146-148). J1 is associated with collagen and other fibrils in muscle and thus may be an extracellular matrix molecule employed in both the central and peripheral nervous systems.

Animals↗

PubMed1986

Molecular specialization of astrocyte processes at nodes of Ranvier in rat optic nerve.

The HNK-1 and L2 monoclonal antibodies are thought to recognize identical or closely associated carbohydrate epitopes on a family of neural plasma membrane glycoproteins, including myelin-associated glycoprotein, the neural cell adhesion molecule, and the L1 and J1 glycoproteins, all of which have been postulated to play a part in mediating cell-cell interactions in the nervous system. We have used these two antibodies in immunofluorescence and immunogold-electron microscopic studies of semithin and ultrathin frozen sections of adult rat optic nerve, respectively, and we show that they bind mainly to astrocyte processes around nodes of Ranvier. Most other elements of the nerve, including astrocyte cell bodies and large astrocytic processes, are not labeled by the antibodies. To our knowledge, this is the first demonstration that perinodal astrocyte processes are biochemically specialized. We provide evidence that one of the HNK-1+/L2+ molecules concentrated around perinodal astrocyte processes is the J1 glycoprotein; our findings, taken together with previously reported observations, suggest that the other known HNK-1+/L2+ molecules are not concentrated on these processes. Since anti-J1 antibodies previously have been shown to inhibit neuron to astrocyte adhesion in vitro, we hypothesize that J1 may play an important part in the axon-glial interactions that presumably are involved in the assembly and/or maintenance of nodes of Ranvier.

Animals↗

PubMed1986

Immunoelectron microscopic localization of neural cell adhesion molecules (L1, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve.

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by pre- and post-embedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non-myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned approximately 1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were L1-negative. At the nodes of Ranvier, the axolemma was also always L1- and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and non-compacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt-Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a non-uniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axon-Schwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N-CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons.

Aging↗

PubMed1986

Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1.

To probe for the role of the L1 cell surface glycoprotein during neurite outgrowth and fasciculation in the early postnatal mouse cerebellar cortex, a microexplant culture system was used. Fasciculation of neurites was reduced in the presence of antigen-binding fragments (Fab) of poly- and monoclonal L1 antibodies, as compared to untreated controls. In addition, speed of neurite outgrowth was enhanced in the presence of antibodies. Migration of cell bodies of small neurons was also significantly increased. Very similar effects on these outgrowth parameters were observed with Fab fragments from poly- and monoclonal neural cell adhesion molecule (N-CAM) antibodies. Antibodies from preimmune sera had no effect. These findings suggest that L1 antigen not only plays a role in adhesion of isolated neural cell bodies and migration of granule cell neurons in the early postnatal mouse cerebellar cortex (Lindner et al., 1983; Rathjen and Schachner, 1984), but also in neurite outgrowth and fasciculation.

Animals↗

PubMed1985

Biosynthesis and membrane topography of the neural cell adhesion molecule L1.

The biosynthesis and membrane topography of the neural cell adhesion molecule L1 have been studied in cerebellar cell cultures by metabolic labeling and immunoprecipitation. Pulse and pulse-chase experiments with [35S]methionine show that L1 is synthesized in its high mol. wt. form, the 200 kd component. The lower mol. wt. components with 40, 80 and 140 K apparent mol. wts. can be generated by proteolysis in intact cellular membranes. Peptide maps generated by protease treatment of L1 isolated from adult mouse brain show that the 80 and 140 kd components are related to the 200 kd component, but not to each other. The 200, 80 and 40 kd components can be biosynthetically phosphorylated. The 140 kd component is not phosphorylated and not released from the surface membrane during tryspinization. The phosphorylated amino acid is serine. In the presence of tunicamycin the 200 kd component is synthesized as a 150 kd protein. Pulse-chase experiments in the presence of tunicamycin indicate that the carbohydrate moieties are predominantly N-glycosidically linked and that the contribution of O-glycosylation is minimal. The carbohydrate moieties are of the complex type as shown by treatment with endoglycosidase H. Since monensin inhibits processing of the carbohydrate moieties, the 200 kd component appears to be transported to the surface membrane via the Golgi apparatus.

Amino Acids↗

PubMed1985

Loop arrays in mouse brain demonstrated with antisera to cytokeratins and monoclonal antibodies to several classes of intermediate filaments: strain differences and developmental expression.

Some monoclonal antibodies raised against mouse brain antigens display a novel loop array apparently localized within the cytoplasm of neurons in fresh frozen sections of adult mouse brain. By indirect immunofluorescence, these loops are detectable in the cerebral cortex, thalamus, brainstem, and are particularly striking in association with pyramidal neurons of the hippocampus. The loops are also seen with polyclonal antibodies to the cytokeratin class of intermediate filaments. The antibodies which react with these loops also react with ependymal cells. Western blot analysis of crude insoluble cytoskeletal components of mouse brain with antibodies of cytokeratins confirm the presence of reactive bands in the range of 40-60 kdalton, appropriate in molecular weight for this class of cytoskeletal filaments. This evidence suggests that the loops share antigenic determinants with non-neural cytokeratins. During development, immunoreactive structures are first seen as small punctate or curvilinear profiles, which change into a loop array at approximately 14 days postnatal age in several mouse strains. However, in 8 of 15 different mouse strains, these immature punctate profiles remain without morphological alteration to loops throughout adult age. The F1 crosses between strains with and without the loops develop loops, but on average they are of smaller size than in the positive parent.

Animals↗

PubMed1985

Histotypic pattern formation in cerebellar reaggregate cultures in the presence of antibodies to L1 cell surface antigen.

Reaggregate cultures of cerebella from 5-day-old C57BL/6J mice were cultured in the presence of Fab fragments of polyclonal antibodies against the cell surface adhesion molecule L1. Light microscopic examination showed that histotypic differentiation, as observed by the appearance of radially oriented glial processes and the sorting out of mature and immature neurons, was not affected by the antibody. Electron microscopic observation showed no effect on the synapse formation and the packing density of fasciculated neurites. These observations show that under the culture conditions used, L1 antibodies do not alter the particular cell interactions investigated in this study.

Animals↗

PubMed1985

Monoclonal cell surface antibodies do not produce short-term effects on electrical properties of mouse oligodendrocytes in culture.

Eleven monoclonal antibodies (O1-O11) directed against the surface of oligodendrocytes were applied individually or in combination during measurement of membrane potential, input resistance and K+-pump activity in explant cultures of mouse spinal cord. Antibody binding to oligodendrocytes was verified by indirect immunofluorescence. None of the antibodies affected the electrical properties studied. On the basis of these observations, it is possible to identify oligodendrocytes immunocytologically prior to electrophysiological characterization.

Animals↗

PubMed1985

Astrocytes support incomplete differentiation of an oligodendrocyte precursor cell.

Glial fibrillary acidic protein-positive astrocytes, but not neurons or fibroblasts, support the differentiation of an oligodendroglial precursor cell expressing O4 antigen and vimentin into an O4 antigen-positive, but vimentin-negative oligodendrocyte. Further maturation into galactocerebroside (O1)-positive oligodendrocytes is, however, not achieved under the culture conditions used, neither in the presence of astrocytes nor neurons.

Animals↗

PubMed1985

Demonstration of immunochemical identity between the nerve growth factor-inducible large external (NILE) glycoprotein and the cell adhesion molecule L1.

The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule L1 were shown to be immunochemically identical. Immunoprecipitation with L1 and NILE antibodies of [3H]fucose-labeled material from culture supernatants and detergent extracts of NGF-treated rat PC12 pheochromocytoma cells yielded comigrating bands by SDS-PAGE. NILE antibodies reacted with immunopurified L1 antigen, but not with N-CAM and other L2 epitope-bearing glycoproteins from adult mouse brain. Finally, by sequential immunoprecipitation from detergent extracts of [35S]methionine-labeled early post-natal cerebellar cell cultures or [3H]fucose-labeled NGF-treated PC12 cells, all immunoreactivity for NILE antibody could be removed by pre-clearing with L1 antibody and vice versa.

Adrenal Gland Neoplasms↗

PubMed1985

Epidermal growth factor does not cross the blood-brain barrier.

To measure the passage of epidermal growth factor (EGF) through the blood-brain barrier (BBB) 125I-labeled EGF was injected intravenously into adult rats. The distribution of 125I-EGF in the blood and cerebrospinal fluid (CSF) was determined over a time period of several hours. Between 2 to 6 h a stable distribution of intact 125I-EGF in CSF was measured to be approximately 1/500 of the blood-borne EGF, an equilibrium value below those obtained by other investigators for BBB-impermeable compounds, such as inulin and bovine serum albumin. Our data indicate that 125I-EGF, although clearly detectable in the CSF, does not cross the BBB at a higher rate or in higher quantities than would be expected from its molecular size.

Animals↗

PubMed1985

Cell type specificity and developmental expression of the L2/HNK-1 epitopes in mouse cerebellum.

The developmental expression of carbohydrate epitopes recognized by monoclonal antibodies HNK-1 and L2 was studied in tissue sections and cultures of mouse cerebellum. At all ages studied the 2 antibodies had identical staining patterns. In cultures of embryonic and early postnatal mice some, but not all cells of all 3 major neural cell types, neurons, astrocytes and oligodendrocytes, but not fibronectin-positive fibroblast-like cells were labeled by the antibodies. The most intensely labeled cells were those with antigenic marker profiles characteristic of immature glia. L2/HNK-1 epitope expression on neuron precursors was detected most convincingly in sections, where the band of migrating granule cell precursors in the external granular layer of the embryo and outer part of the external granular layer at early postnatal ages were strongly labeled by the antibodies. In sections of adult cerebella an overall reduced, but detectable level of L2/HNK-1 epitope expression was seen in all layers, but slightly more prominently in granular layer and white matter. At embryonic ages ventricular cells did not express detectable levels of epitopes. Most of the present data support the notion that the L2/HNK-1 epitopes are most strongly expressed on cell types with properties of glial and neuronal precursor cells.

Animals↗

PubMed1985

Selective expression of the 180-kD component of the neural cell adhesion molecule N-CAM during development.

The rodent neural cell adhesion molecule (N-CAM) consists of three glycoprotein chains of 180, 140, and 120 kD in their adult forms. Although the proportions of the three components are known to change during development and differ between brain regions, their individual distribution and function are unknown. Here we report studies carried out with a monoclonal antibody that specifically recognizes the 180-kD component of mouse N-CAM (N-CAM180) in its highly sialylated embryonic and less glycosylated adult forms. In primary cerebellar cell cultures, N-CAM180 antibody reacts intracellularly with all types of neural cells including astrocytes, oligodendrocytes, and neurons. During cerebellar, telencephalic, and retinal development N-CAM180 is detectable by indirect immunohistology in differentiated neural cells, but, in contrast to total N-CAM, not in their proliferating precursors in the ventricular zone and primordial and early postnatal external granular layer. In monolayer cultures of C1300 neuroblastoma cells, N-CAM180 appears by immunofluorescence more concentrated at contact points between adjacent cells, while N-CAM comprising the 180- and 140-kD component shows a more uniform distribution at the plasma membrane. Treatment of neuroblastoma cells with dimethylsulfoxide, which promotes differentiation, induces a shift toward the predominant expression of N-CAM180. These observations support the notion that N-CAM180 is expressed selectively in more differentiated neural cells and suggest a differential role of N-CAM180 in the stabilization of cell contacts.

Aging↗

PubMed1985

Expression of the neural cell adhesion molecules L1 and N-CAM and their common carbohydrate epitope L2/HNK-1 during development and after transection of the mouse sciatic nerve.

The expression of the neural cell adhesion molecules L1 and N-CAM and of their shared carbohydrate epitope L2/HNK-1 was studied during the development and after the transection of mouse sciatic nerves. During development, L1 and N-CAM were detectable on most, if not all, Schwann cells at embryonic day 17, the earliest stage tested. With increasing age, the immunoreactivity was reduced being confined to non-myelinating Schwann cells by post-natal day 10, at which stage the staining pattern resembled that seen in adult sciatic nerves. Double-immunolabelling experiments revealed a complete overlap between L1 and N-CAM antibodies. The L2/HNK-1 epitope was not detectable in developing sciatic nerves until the end of the 2nd post-natal week, when it appeared to be associated with the outer profiles of thick myelin sheets, as also seen in adult sciatic nerves. Three days after the transection of adult sciatic nerves, L1 antigen and N-CAM was detectable in more Schwann cells in the distal nerve end than in untreated control nerves. The peak level of the reappearance of L1 antigen and N-CAM in Schwann cells occurred between 2 and 4 weeks after transection. The reduction of L1-antigen expression to its normal adult level took more than a year, thus recapitulating normal development, but on a more protracted time scale. Similarly, the L2/HNK-1 epitope remained undetectable until the transected nerve had returned to its normal state of myelination, i.e. approximately 1 year after transection.

Aging↗

PubMed1985

Glycosaminoglycans of rat cerebellum: I. Quantitative analysis of the main constituents at postnatal day 6.

Isolated glycosaminoglycans (GAGs) were quantified biochemically in the cerebella of 6-day-old rats. 14C-Labeled hyaluronic acid (HA) and chondroitin-4-sulfate (C-4-S), added prior to isolation of GAGs from tissue, served as internal standards to allow correction for unknown losses during the purification procedure and exact quantification of GAGs in the intact tissue. Three main constituents--HA, chondroitin sulfate (CS), and heparan sulfate (HS)--were found at concentrations of 1.82, 1.52, and 0.76 micrograms/mg protein amounting to 44%, 37%, and 19% of the total GAG fraction, respectively. Incorporation of [3H]glucosamine precursor into GAGs was higher for HS (56% of incorporated precursor) and lower for HA (29%) and CS (15%). The specific activities of individual GAGs were 64.7 nCi/micrograms for HS, 14.2 for HA, and 8.3 for CS.

Acetic Anhydrides↗

PubMed1985

Glycosaminoglycans of rat cerebellum: II. A developmental study.

Total and individual glycosaminoglycans (GAGs) were determined in rat cerebellum in tissue explants at various postnatal ages. The major constituents of GAGs were chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). Dermatan sulfate (DS) and keratan sulfate (KS) could not be detected and therefore each amounts to less than 5% of all GAGs at all ages studied. HA was the prominent GAG during postnatal development and only a minor constituent at adult ages, whereas CS was the predominant GAG in adulthood. HS remained relatively constant throughout development. The incorporation of [3H]glucosamine into individual GAGs was highest for HS at postnatal day 6, whereas HA showed intermediate and CS the lowest levels of incorporation during the first postnatal week. All major GAGs showed the lowest incorporation values at adult ages.

Age Factors↗

PubMed1985

Pharmacological properties of gamma-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in cultured astrocytes.

Differentiated glial fibrillary acidic protein-positive astrocytes in homogeneous cultures of early postnatal rat cerebral hemispheres respond by membrane depolarization to gamma-aminobutyric acid (GABA), glutamate, and aspartate with a threshold concentration of approximately 10(-5) M. The GABA-induced depolarization is antagonized by two blockers of the neuronal GABAA receptor, picrotoxin and bicuculline, but is not affected by the uptake blockers beta-alanine or nipecotic acid. An agonist of the GABAA receptor, muscimol, produces a dose-response curve similar to that of GABA, whereas the agonist of the GABAB receptor, baclofen, did not alter the membrane potential. When repetitive pulses of GABA are given to one cell, its responsiveness depends on the time interval between pulses. Within 30 sec after termination of the first pulse the cell remains unresponsive to the second pulse. With increased time intervals between the pulses, reactivity toward GABA recovers. Five minutes after the first pulse the cell regains 75% of its initial depolarization peak. Aspartate results in a depolarization similar in size and time course to that induced by glutamate. The glutamate agonists, quisqualate and ibotenate, and kainate are less potent than glutamate. N-Methyl-D-aspartate has no effect on the membrane potential of astrocytes. The pharmacological features of the glutamate response are therefore similar to those of the receptor mediating neuronal glutamate transport.

Animals↗

PubMed1984

Aspartate, glutamate and gamma-aminobutyric acid depolarize cultured astrocytes.

Cultures of differentiated, glial fibrillary acidic protein-positive astrocytes from early postnatal rat cerebral hemispheres respond with depolarization of 2-36 mV to glutamate, gamma-aminobutyric acid (GABA) and aspartate but not to glycine or taurine. While GABA resulted in a transient depolarization, the effect of glutamate and aspartate persisted during the application. Since neurons were not present in these cultures a contribution of transmitter-mediated K+ release from adjacent neurons could be excluded. The depolarization triggered by these neurotransmitters is therefore an intrinsic reaction of astrocytes.

Animals↗