Rainer Probstmeier

Binding Properties of the Neural Cell Adhesion Molecule to Different Components of the Extracellular Matrix

Abstract: A soluble form of the neural cell adhesion molecule (N‐CAM) was obtained from 100,000‐g supernatants of crude brain membrane fractions by incubation for 2 h at 37°C. The isolated N‐CAM, consisting of one polypeptide chain with a molecular mass of 110 kilodaltons (N‐CAM 110), was studied for its binding specificity to different components of the extracellular matrix (ECM). N‐CAM 110 bound to different types of collagen (collagen types I‐VI and IX). The binding efficiency was dependent on salt concentration and could be called specific according to the following criteria: (a) Binding showed substrate specificity (binding to collagens, but not to other ECM components, such as laminin or fi‐bronectin). (b) Binding of N‐CAM 110 to heat‐denatured collagens was absent or substantially reduced, (c) Binding was saturable (Scatchard plot analyses were linear with KD values in the range of 9.3‐2.0 ± 10−9M, depending on ihe collagen type and buffer conditions). Binding of N‐CAM 110 to collagens could be prevented in a concentration‐dependent manner by the glycosaminoglycans heparin and chondroitin sulfate. N‐CAM 110 also interacted with immobilized heparin, and this interaction could be prevented by heparin and chondroitin sulfate. Thus, in addition to its role in cell‐cell adhesion, N‐CAM is a binding partner for different ECM components, an observation suggesting that it also serves as a substrate adhesion molecule in vivo

Experimental Axonal Injury Triggers Interleukin-6 mRNA, Protein Synthesis and Release into Cerebrospinal Fluid:

Diffuse axonal injury is a frequent pathologic sequel of head trauma, which, despite its devastating consequences for the patients, remains to be fully elucidated. Here we studied the release of interleukin-6 (IL-6) into CSF and serum, as well as the expression of IL-6 messenger ribonucleic acid (mRNA) and protein in a weight drop model of axonal injury in the rat. The IL-6 activity was elevated in CSF within 1 hour and peaked between 2 and 4 hours, reaching maximal values of 82,108 pg/mL, and returned to control values after 24 hours. In serum, the levels of IL-6 remained below increased CSF levels and did not exceed 393 pg/mL. In situ hybridization demonstrated augmented IL-6 mRNA expression in several regions including cortical pyramidal cells, neurons in thalamic nuclei, and macrophages in the basal subarachnoid spaces. A weak constitutive expression of IL-6 protein was shown by immunohistochemical study in control brain. After injury, IL-6 increased at 1 hour and remained elevated through the first 24 hours, returning to normal afterward. Most cells producing IL-6 were cortical, thalamic, and hippocampal neurons as confirmed by staining for the neuronal marker NeuN. These results extend our previous studies showing IL-6 production in the cerebrospinal fluid of patients with severe head trauma and demonstrate that neurons are the main source of IL-6 after experimental axonal injury.

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.

The yin and yang of tenascin-R in CNS development and pathology.

An important biological consequence of the initial interactions between the cell surface and its extracellular environment is the diversity of cellular responses ranging from overt repulsion or avoidance reaction to stable adhesion or final positioning. It is now evident that positive and negative guiding mechanisms are equally relevant to normal pattern formation during development and decisive for the outcome of a regenerative process. In this context, the present review summarizes the knowledge about the extracellular matrix glycoprotein tenascin-R, a member of the tenascin gene family. In contrast to all other known family members, tenascin-R is exclusively expressed in the central nervous system of vertebrates by oligodendrocytes and neuronal subsets at later developmental stages and in adulthood. We focus on the glycoproteins structure, tissue distribution and functional implications in the molecular control of axon targeting, neural cell adhesion, migration and differentiation during nervous system morphogenesis and pathology.

Galectin-3 promotes neural cell adhesion and neurite growth

Galectin‐3 is a member of the galectin family and belongs to a group of soluble β‐galactoside‐binding animal lectins. The molecule is expressed by neural and nonneural cells intra‐ (cytoplasm and nucleus) as well as extra‐cellularly (plasma membrane and extracellular space). By using an in vitro cell‐substratum adhesion assay, we have addressed the question whether galectin‐3 present in the extracellular milieu may support the adhesion and/or neurite outgrowth of neural cells in a manner analogous to cell adhesion molecules. Galectin‐3 was immobilized as a substratum and various cell types, N2A (neuroblastoma), PC12 (pheochromocytoma), and TSC (transformed Schwann cells) cell lines, neural cells from early postnatal mouse cerebellum, and dorsal root ganglion neurons from newborn mice were allowed to adhere to the lectin. Here we show that all cell types studied specifically adhered to galectin‐3 by the following criteria: 1) the number of adherent cells was dependent on the galectin‐3 concentration used for coating; 2) adhesion of cells to galectin‐3, but not to collagen type I or laminin was inhibited by polyclonal antibodies to galectin‐3; 3) upon addition of asialofetuin (a polyvalent carrier of terminal β‐galactosides) to the cell suspension prior to the adhesion assay, cell adhesion to galectin‐3 was inhibited in a dose‐dependent manner; and 4) cell adhesion to galectin‐3 was abolished by treatment of cells with endo‐β‐galactosidase. In addition, the adhesion of dorsal root ganglion neurons to galectin‐3 could be inhibited by lactose. Notably, substratum‐bound galectin‐3 promoted the outgrowth of neurites from dorsal root ganglia explants and this neurite outgrowth promoting activity could be inhibited by polyclonal antibodies to galectin‐3. J. Neurosci. Res. 54:639‐654, 1998.

Galectin‐3 is upregulated in microglial cells in response to ischemic brain lesions, but not to facial nerve axotomy

We have recently demonstrated that the β‐galactoside‐specific lectin galectin‐3 is expressed by microglial cells in vitro, but not by normal resting microglia in vivo. In the present study, we have analyzed the expression of galectin‐3 by microglia under traumatic conditions in vivo using two experimental rat models which substantially differ in the severity of lesion related to a breakdown of the blood‐brain barrier (BBB) and the occurrence of inflammatory processes. These two features are absent after peripheral nerve lesion and present after cerebral ischemia. Here we show that, following facial nerve axotomy under conditions allowing (nerve anastomosis) or not subsequent regeneration (nerve resection), galectin‐3 is not expressed by microglia in the corresponding facial nucleus 1–112 days after lesion. Galectin‐3 is also absent in microglia at sites of a defective BBB in the normal brain, such as the circumventricular organs. Following experimental ischemia (i.e., permanent occlusion of the middle cerebral artery), in contrast, galectin‐3 becomes strongly expressed by activated microglia as early as 48 hours after trauma, as determined by immunohistochemistry and Western blot analysis. Our findings suggest that the expression of galectin‐3 by microglia in vivo correlates with the state of microglial activation. J. Neurosci. Res. 61:430–435, 2000.

Runx2 Is Expressed in Human Glioma Cells and Mediates the Expression of Galectin-3

Runx2 is a member of the Runx family of transcription factors (Runx1–3) with a restricted expression pattern. It has so far been detected predominantly in skeletal tissues where, inter alia, it regulates the expression of the β‐galactoside‐specific lectin galectin‐3. Here we show that, in contrast to Runx3, Runx1 and Runx2 are expressed in a variety of human glioma cells. Runx2 expression pattern in these cells correlated completely with that of galectin‐3, but not with that of other galectins. A similar correlation in the expression pattern of galectin‐3 and Runx2 transcripts was detected in distinct types of 70 primary neural tumors, such as glioblastoma multiforme, but not in others, such as gangliocytomas. In glioma cells, Runx2 is directly involved in the regulation of galectin‐3 expression, as shown by RNAi and transcription factor binding assays demonstrating that Runx2 interacts with a Runx2‐binding motif present in the human galectin‐3 promoter. Knockdown of Runx2 was thus accompanied by a reduction of both galectin‐3 mRNA and protein levels by at least 50%, dependent on the glial tumor cell line tested. Reverse transcriptase–polymerase chain reaction analyses, aimed at finding other potential target genes of Runx2 in glial tumor cells, revealed the presence of bone sialoprotein, osteocalcin, osteopontin, and osteoprotegerin. However, their expression patterns only partially overlap with that of Runx2. These data suggest a functional contribution of Runx‐2‐regulated galectin‐3 expression to glial tumor malignancy.

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.

Homophilic binding properties of galectin-3 : Involvement of the carbohydrate recognition domain

Abstract: Galectin‐3, an animal lectin specific for β‐galactosides, is composed of three different domains. The N‐terminal half of the molecule (N domain) consists of a short N‐terminal segment followed by glycine‐, proline‐, and tyrosine‐rich tandem repeats. The C‐terminal domain (C domain) harbors the carbohydrate recognition domain homologous to other members of the galectin family of lectins. Galectin‐3 aggregates in solution, and participation of the N domain of the molecule in this process has already been demonstrated. Using a solid‐phase radioligand binding assay, which allows the direct analysis of galectin‐3 self‐association, here we provide evidence that the carbohydrate recognition domain of the lectin is involved in carbohydrate‐dependent homophilic interactions: (a) Radiolabeled galectin‐3 binds to immobilized galectin‐3, and the addition of unlabeled galectin‐3 in solution increases the rate of binding of radiolabeled lectin; (b) binding of radiolabeled galectin‐3 to immobilized galectin‐3 is inhibited by the C domain; (c) binding of radiolabeled galectin‐3 to immobilized galectin‐3 or the C domain is inhibited by lactose but not by sucrose; and (d) the radiolabeled C domain does not bind to immobilized C domain. Taken together, these data suggest that in addition to the N domain, the homophilic interactions of galectin‐3 are mediated by the C domain.

Tenascin‐R interferes with integrin‐dependent oligodendrocyte precursor cell adhesion by a ganglioside‐mediated signalling mechanism

Oligodendrocyte (OL) lineage progression is characterized by the transient expression of the disialoganglioside GD3 by OL precursor (preOL) cells followed by the sequential expression of myelin‐specific lipids and proteins. Whereas GD3+ preOLs are highly motile cells, the migratory capacity of OLs committed to terminal differentiation is strongly reduced, and we have recently shown that the extracellular matrix protein tenascin‐R (TN‐R) promotes the stable adhesion and differentiation of O4+ OLs by a sulphatide‐mediated autocrine mechanism (O4 is a monoclonal antibody recognizing sulphatides/seminolipids expressed by OLs and in myelin). Using culture conditions that allow the isolation of mouse OLs at distinct lineage stages, here we demonstrate that TN‐R is antiadhesive for GD3+ preOLs and inhibits their integrin‐dependent adhesion to fibronectin (FN) by a disialoganglioside‐mediated signalling mechanism affecting the tyrosine phosphorylation of the focal adhesion kinase. This responsive mechanism appears to be common to various cell types expressing disialogangliosides as: (i) disialogangliosides interfered with the inhibition of cell adhesion of different neural and non‐neural cells on substrata containing TN‐R and FN or RGD‐containing FN fragments. TN‐R interacted specifically with disialoganglioside‐expressing cells or immobilized gangliosides, and ganglioside treatment of TN‐R substrata resulted in a delayed preOL cell detachment as a function of time. We conclude that OL response to one and the same signal in the extracellular matrix critically depends on the molecular repertoire expressed by OLs at different lineage stages and could thus define their final positioning.