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M Schachner

Publications and source records attributed to M Schachner.

At least 379 records · Page 21Linked to original sources

Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration.

Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.

Adenosine Triphosphatases↗

The novel carbohydrate epitope L3 is shared by some neural cell adhesion molecules.

The monoclonal L3 antibody reacts with an N-glycosidically linked carbohydrate structure on at least nine glycoproteins of adult mouse brain. Three out of the L3 epitope-carrying glycoproteins could be identified as the neural cell adhesion molecules L1 and myelin-associated glycoprotein, and the novel adhesion molecule on glia. Expression of the L3 carbohydrate epitope is regulated independently of the protein backbone of these three glycoproteins. Based on the observation that out of three functionally characterized L3 epitope-carrying glycoproteins three fulfill the operational definition of an adhesion molecule, we would like to suggest that they form a new family of adhesion molecules that is distinct from the L2/HNK-1 carbohydrate epitope family of neural cell adhesion molecules. Interestingly, some members in each family appear to be unique to one family while other members belong to the two families.

Adenosine Triphosphatases↗

Immunoelectron microscopic localization of the neural cell adhesion molecules L1 and N-CAM during postnatal development of the mouse cerebellum.

The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre- and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM180) stain only the postmigratory granule cell bodies supporting the notion that N-CAM180, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAM180 is only transiently expressed on Purkinje cell dendrites. N-CAM is present in synapses on both pre- and post-synaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes. These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.

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Myelin-associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules, is involved in neuron-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction.

A monoclonal antibody to the myelin-associated glycoprotein (MAG) was prepared and characterized to probe for the involvement of MAG in cell surface interactions among neural cells in vitro. The antibody reacts specifically with oligodendrocyte cell surface and myelin-rich brain regions as expected from previous investigations. Not all O4 antigen-positive oligodendrocytes express MAG in vitro. Fab fragments of the antibody interfere with neuron to oligodendrocyte and oligodendrocyte to oligodendrocyte adhesion, but not with oligodendrocyte to astrocyte adhesion. MAG-containing liposomes bind to the cell surfaces of the appropriate target cells by a mechanism that is specifically inhibitable by Fab fragments of monoclonal MAG antibodies, demonstrating that MAG is a neural cell adhesion molecule.

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Release of the 120 kDa component of the mouse neural cell adhesion molecule N-CAM from cell surfaces by phosphatidylinositol-specific phospholipase C.

To study the membrane anchoring of the 120 kDa component of the neural cell adhesion molecule N-CAM, the smallest form lacking a transmembrane domain, cultured mouse neural cells were treated with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. When live cultures of astrocytes and neurons are treated with phosphatidylinositol-specific phospholipase C, N-CAM120 is released into the supernatant. Under these conditions N-CAM140 and N-CAM180 are not released. Phospholipase C from Bacillus cereus or Clostridium perfringens does not release N-CAM120. The embryonic form of N-CAM on astrocytes migrating as a broad band between 120 and 180 kDa is also partially released by phosphatidylinositol-specific phospholipase C as a band migrating between 120 and 160 kDa. These observations suggest novel mechanisms in regulation of N-CAM120 expression on the cell surface and in modulation of N-CAM-mediated cell adhesion.

Aging↗

Molecular specificity of L2 monoclonal antibodies that bind to carbohydrate determinants of neural cell adhesion molecules and their resemblance to other monoclonal antibodies recognizing the myelin-associated glycoprotein.

L2 monoclonal antibodies and HNK-1 have been shown to bind to related carbohydrate determinants in the myelin-associated glycoprotein (MAG) and several adhesion molecules of the nervous system including neural cell adhesion molecule (N-CAM), L1 and J1. It is shown here that MAG is the principal component in human white matter binding the L2 antibodies, but the most prominent antigens with the L2 epitopes in human gray matter are of higher Mr. It is also shown that the L2 antibodies resemble HNK-1 in binding to some 19-28 kDa glycoproteins and some sulfated, glucuronic acid-containing sphingoglycolipids of the peripheral nervous system (PNS). In addition, monoclonal and polyclonal antibodies raised to human MAG are shown to cross react with bovine N-CAM due to the presence of common carbohydrate constituents. The results further emphasize the shared antigenicity between MAG, N-CAM and other adhesion molecules. In addition, they demonstrate that the L2 antibodies belong to a family of monoclonal antibodies (including HNK-1, human IgM paraproteins associated with neuropathy, and others) that are characterized by reactivity against carbohydrate determinants shared by human MAG, the 19-28 kDa glycoproteins of the PNS and the sulfated, glucuronic acid-containing sphingoglycolipids of the PNS.

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Experimental modification of postnatal cerebellar granule cell migration in vitro.

Histotypic migration of [3H]thymidine pulse-labeled granule cell neurons in cerebellar folium explants was monitored in the presence of antibodies to cell adhesion molecules and quantified by automatic image analysis. When explants were cultured in the presence of monovalent antibody fragments to cell adhesion molecules L1 and N-CAM, an inhibition of cell migration of 33.3 +/- 4.4% and 13.9 +/- 2.1%, respectively, was observed. In the presence of an equimolar mixture of monovalent antibody fragments to L1 antigen and N-CAM no additive effects in inhibition of cell migration were seen. Antibodies to the L2 carbohydrate epitope which is common to L1, N-CAM and other cell surface glycoproteins showed a similarly small effect on cell migration as antibodies to N-CAM. Monoclonal antibodies to cell surface antigen M2 and polyclonal antibodies to mouse liver membranes reacting with the surface of all cerebellar cell types did not alter the migratory behavior of granule cells. Cultivation of explants in the presence of neuraminidase, ganglioside binding toxins, as well as glycosaminoglycans and glycosaminoglycan degrading enzymes, also did not modify the extent of cell migration under the culture conditions used.

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Molecular association of two neural cell adhesion molecules within the surface membrane of cultured mouse neuroblastoma cells.

The neural cell adhesion molecule L1 can be induced by antibodies in indirect immunofluorescence procedures to co-redistribute on the surface membrane of cultured mouse neuroblastoma cells with the 180 kDa component of N-CAM, but not with the 140 kDa component of N-CAM, the H-2 histocompatibility antigen or antigens recognized by polyspecific antibodies to mouse liver membranes. These observations indicate a differential and close molecular association between L1 and the N-CAM component with the larger intracellular domain.

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Dissociated cells of foetal rat pallium grown in culture medium supplemented with noradrenaline: effects on the expression of neuron-specific enolase and cell adhesion molecule L1.

The possible influence of noradrenaline (NA) upon cell differentiation has been studied by comparing NA-supplemented cultures of foetal pallial cells with control cultures grown in normal medium. Two days after plating, the cultures were processed for immunocytochemical detection of either an adhesion molecule and marker of early stages of neuronal differentiation (L1) or a marker expressed at relatively late stages (gamma-enolase). In both cases, the NA supplement reduced the expression of the antigen. The effects were more clear-cut for the late than for the early marker. In conclusion, the NA supplement to the culture medium, in our model, seemed to have a 'differentiation regulating' rather than a 'neurotrophic' function sensu stricto. It remains to be clarified, however, to which extent this finding can be generalized to in vivo situations.

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Reactivity with neural cell adhesion molecules in sera from patients with demyelinating diseases.

Sera from 5 patients with IgM gammopathy and peripheral neuropathy reacted strongly by a sensitive immunospot enzyme-linked immunosorbent assay test with the myelin-associated glycoprotein (MAG) purified from human and bovine brains and the novel neural cell adhesion molecule J1 and associated glycoproteins from human brain. Two other members of the L2/HNK-1 family of cell adhesion molecules, the L1 glycoprotein and the neural cell adhesion molecule N-CAM, are not recognized significantly more strongly by these sera than by sera from patients with multiple sclerosis. Sera from patients with Guillain-Barré syndrome and amyotrophic lateral sclerosis and from healthy humans did not react with any of the glycoproteins tested at the serum concentrations used.

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Single voltage-dependent K+-channels in cultured astrocytes.

The open-occupation time of about 35% of the K+ channels in astrocytes increases strongly with depolarization. The kinetics of one of three different types of voltage-dependent channels (present in about 10% of all records) have been studied in detail. Openings can be fitted with one exponential, while a sum of three exponentials is necessary to fit the distributions of closures. A reaction scheme of one open and three closed states is necessary to describe the kinetic reactions of this channel. Voltage dependence is expressed by a strong increase of burst duration with depolarization. At resting potential (-70 mV), the channel contributes little to the resting membrane potential. Its steady-state open probability (P0) is 0.058. When the cell is depolarized, P0 can increase up to 0.96. Functionally, such channels could contribute to the potassium clearance by enhancing the effect of 'spatial buffering'.

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Epidermal growth factor is not detectable in developing and adult rodent brain by a sensitive double-site enzyme immunoassay.

A highly sensitive double-site enzyme immunoassay for epidermal growth factor (EGF) was used to quantify EGF concentrations in brain and cerebrospinal fluid of early postnatal and adult mice and rats. EGF was not detectable under any condition at sensitivity levels of 0.06 ng/g wet wt. These observations support the notion that EGF receptors on astrocytes are triggered by other growth factors than EGF.

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Conversion of embryonic to adult form of the neural cell adhesion molecule (N-CAM) does not correlate with pre- and postmigratory states of mouse cerebellar granule neurons.

The migration of postmitotic granule neurons from the external to the internal granular layer along the radial processes of Bergmann glia is an outstanding event in the development of the early postnatal mouse cerebellar cortex. It has been hypothesized that the conversion of the embryonic to the adult form of the neural cell adhesion molecule (N-CAM) is important for this migration since this conversion coincides with the time of migration [2, 10]. To investigate this issue we have examined the molecular forms of N-CAM by Western blot analysis in tissue obtained from hand-dissected cerebellar layers at several stages of early postnatal development. Here we report that there is no temporal difference between internal and external granular layers in shifting from exclusive expression of the embryonic form of N-CAM at postnatal day 1 to predominance of the adult form at day 17. White matter shows a delay in the conversion to the adult form which is completed only after day 17. We conclude that the transition from embryonic to adult form of N-CAM is not a prerequisite for granule neuron migration.

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Distribution of the adhesion molecules N-CAM and L1 on peripheral neurons and glia in adult rats.

There is considerable evidence that the cell surface glycoproteins N-CAM and L1 are important mediators of cell-cell adhesion in the nervous system, at least during development. Numerous studies have been devoted to the molecular properties of these proteins and their adhesion role in embryonic and early postnatal development. Much less is known about their importance in mature tissues. A rigorous and comprehensive description of the cell distribution of these molecules in the adult nervous system would clearly form a useful baseline for functional and biochemical studies. In the present work we have addressed this issue and studied the distribution of N-CAM and L1 throughout adult, as opposed to developing, rat peripheral nervous tissue. Particular attention was paid to the ganglia of the enteric nervous system, since adhesion mechanisms within these ganglia are likely to be placed under unusual demands. We report, for the first time, the presence of N-CAM and L1 on mature sensory, sympathetic and enteric neurons in adult rats. Thus, immunostaining of cell suspensions or short-term cultures showed N-CAM and L1 surface labelling on sympathetic and both large and small dorsal root sensory neurons. Both antigens were also present on the surface of enteric neurons in cultures prepared from 10-day-old rats and neonatal guinea pigs. Immunostaining of sections of enteric ganglia from adults indicated that both molecules were also expressed by mature enteric neurons. In sections of mature sciatic nerve neither N-CAM nor L1 immunoreactivity were detected at the site where the plasma membrane of myelinated axons meets the ad-axonal plasma membrane of the myelin-forming Schwann cell. Thus, both N-CAM and L1 were detected on all major classes of peripheral neurons, while their levels in the plasma membrane of myelinated axons may be significantly down-regulated. Similarly, both N-CAM and L1 were present on all major classes of non-myelin-forming peripheral glia in adult rats. This includes the enteric glial cells of the myenteric ganglia, non-myelin-forming Schwann cells in the sciatic nerve, sympathetic trunk and fine autonomic nerves in the gut wall, and the satellite glial cells of sympathetic and dorsal root sensory ganglia. In contrast, myelin-forming Schwann cells did not express detectable levels of N-CAM and only very low levels of L1, which was mainly located near the nodes of Ranvier.(ABSTRACT TRUNCATED AT 400 WORDS)

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Comparison of astrocytic morphology, proliferation, marker profile and response to neurons in wild-type and weaver mutant mouse cerebella in culture.

In serum-free monolayer cultures of early postnatal weaver (wv/wv) cerebellum granule neurons show decreased attachment, survival and neurite outgrowth when compared to wild-type (+/+) littermate cultures. wv/wv Astrocytes display a more epithelioid morphology and altered proliferation. However, both morphology and proliferation of wv/wv astrocytes were reversed to a normal phenotype by addition of purified small neurons from early postnatal cerebella from +/+ animals. Attachment of +/+ neurons to wv/wv astrocytes was not significantly different from that of +/+ astrocytes and antigenic marker profiles of wv/wv and +/+ astrocytes differed only slightly. Attempts failed to revert the abnormal wv/wv phenotype in neurons by addition of gangliosides, triiodothyronine T3, prostaglandin A2, medium containing 1% horse serum, conditioned medium from +/+ cerebellar cultures, or by cocultivation with +/+ astrocytes. We would like to suggest that the primary defect of the wv/wv mutation is predominantly an abnormality in granule cell neurons, but not of the vast majority of astrocytes.

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Immunocytological localization of cell adhesion molecules L1 and N-CAM and the shared carbohydrate epitope L2 during development of the mouse neocortex.

The expression of the two adhesion molecules L1 and N-CAM and their shared carbohydrate epitope recognized by monoclonal antibody L2, was studied during development of the embryonic mouse neocortex by immunohistology at light- and electron-microscopic levels between embryonic days 9 and 18. Throughout this time period N-CAM is expressed in all layers of the telencephalic anlage. L1 antigen shows a more restricted expression than N-CAM. It is not detectable at day 9. From day 10 onward it is expressed on young neurons in the marginal zone, but not in the ventricular layer. At embryonic day 13 L1 antigen appears also in the intermediate zone on afferent fibers from subcortical structures and on migrating neurons. Neuronal cell bodies in the cortical plate and subplate express L1 antigen only transiently on embryonic days 13-16. These observations suggest that L1 antigen does not play a prominent role in the initiation of neuronal migration in the ventricular zone, but could be functional during later stages of migration and in the aggregation of neuronal cell bodies at their final position in the cortical plate. The L2 epitope also shows a more restricted expression than N-CAM during the time period studied. Similar to L1 antigen, it first appears at embryonic day 10 in the marginal zone and remains undetectable in the ventricular layer also at later stages. In the marginal zone the L2 epitope is strongly expressed on neuroepithelial endfeet at the basal lamina. The basal lamina itself is L2 epitope-negative. From embryonic day 10 onward the L2 epitope is most strongly expressed in the marginal zone and subplate and more weakly in the cortical plate and intermediate zone. In the subplate it is not only associated with the surface membrane, but also with the extracellular matrix. These observations support previous biochemical data which show that the L2 epitope is not present on all N-CAM molecules of the embryonic or adult forms and suggest that the independent regulation or L2 epitope expression may have functional implications during development.

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Neuronal influence on antigenic marker profile, cell shape and proliferation of cultured astrocytes obtained by microdissection of distinct layers from the early postnatal mouse cerebellum.

To study the cellular heterogeneity of astrocytes from early postnatal mouse cerebellum in culture, Bergmann glia were enriched by hand-dissection of Purkinje, molecular and external granular layers ('outer' layer) and fibrous astrocytes of white matter and deep cerebellar nuclei ('inner' layer). Both populations of GFA protein and vimentin-positive astrocytes express N-CAM and the L2/HNK-1 epitope, but not tetanus toxin receptors or A2B5 antigen, at levels detectable by indirect immunofluorescence procedures. The two astrocyte populations are thus indistinguishable from each other. Expression of tetanus toxin receptors and A2B5 antigen in these astrocytes can, however, be induced by removal of neurons. The expression of tetanus toxin receptors is again reduced by readdition of purified populations of small cerebellar neurons. Morphology and proliferation of astrocytes from both layers is also dependent on the presence of neurons: removal of neurons leads to an epithelioid, rather than star-shaped morphology and a severalfold increase in proliferation. Readdition of neurons induces astrocytes to return to their star-shaped morphology. Epidermal growth factor increases proliferation in both populations of astrocytes. We conclude that neither antigenic marker profile, morphology nor proliferative responses serve to distinguish between enriched Bergmann glia and enriched fibrous astrocytes.

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Modulation of granule cell migration by a glia-derived protein.

Cultured explants from early postnatal mouse cerebellum were used to examine the influence of a 43-kDa glia-derived neurite-promoting factor (GdNPF) on the migration of [3H]thymidine-labeled granule cell neurons. GdNPF, which is a potent serine protease inhibitor, significantly reduced the extent of granule cell migration in a dose-dependent manner. This effect could be neutralized by addition of thrombin, which binds GdNPF. Other protease inhibitors such as aprotinin, hirudin, soybean trypsin inhibitor, leupeptin, 6-aminocaproic acid, and D-Phe-Pro-ArgCH2Cl do not show this inhibitory effect. These results demonstrate that a glia-derived protein can regulate the migration of postmitotic neurons, an important cellular event in the development of the nervous system.

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