Penka Pesheva

Versican Is Upregulated in CNS Injury and Is a Product of Oligodendrocyte Lineage Cells

Chondroitin sulfate proteoglycan (CS-PG) expression is increased in response to CNS injury and limits the capacity for axonal regeneration. Previously we have shown that neurocan is one of the CS-PGs that is upregulated (Asher et al., 2000). Here we show that another member of the aggrecan family, versican, is also upregulated in response to CNS injury. Labeling of frozen sections 7 d after a unilateral knife lesion to the cerebral cortex revealed a clear increase in versican immunoreactivity around the lesion. Western blot analysis of extracts prepared from injured and uninjured tissue also revealed considerably more versican in the injured tissue extract. In vitrostudies revealed versican to be a product of oligodendrocyte lineage cells (OLCs). Labeling was seen between the late A2B5-positive stage and the O1-positive pre-oligodendrocyte stage. Neither immature, bipolar A2B5-positive cells, nor differentiated, myelin-forming oligodendrocytes were labeled. The amount of versican in conditioned medium increased as these cells differentiated. Versican and tenascin-R colocalized in OLCs, and coimmunoprecipitation indicated that the two exist as a complex in oligodendrocyte-conditioned medium. Treatment of pre-oligodendrocytes with hyaluronidase led to the release of versican, indicating that its retention at the cell surface is dependent on hyaluronate (HA). In rat brain, approximately half of the versican is bound to hyaluronate. We also provide evidence of a role for CS-PGs in the axon growth-inhibitory properties of oligodendrocytes. Because large numbers of OLCs are recruited to CNS lesions, these results suggest that OLC-derived versican contributes to the inhospitable environment of the injured CNS.

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.

MURINE MICROGLIAL CELLS EXPRESS FUNCTIONALLY ACTIVE GALECTIN-3 IN VITRO

In the present study we have analyzed the expression of galectin‐3, a β‐galactoside‐specific soluble animal lectin, by microglial cells in vitro. In enriched microglial cell cultures derived from neonatal mouse brain after 2 to 3 weeks in vitro, almost all microglial cells expressed galectin‐3 intracellularly and about 90% expressed the molecule on the cell surface. Western blot analyses of lysates from microglial cells using galectin‐3‐specific antibodies revealed a single band with an apparent molecular weight of 29 kD. The carbohydrate recognition domain of microglia‐derived galectin‐3 was functional as the molecule could be affinity purified on lactose‐agarose. Upon an incubation with lactose‐, but not with sucrose‐containing buffers the amount of cell surface expressed galectin‐3 was strongly reduced, suggesting that the molecule appears to be associated with the plasma membrane via its carbohydrate recognition domain. The total amount as well as the portion of cell surface expressed galectin‐3 increased upon treatment with granulocytemacrophage colony‐stimulating factor. Our findings suggest that galectin‐3 expression is subject to regulation by growth factors supposed to be involved in the cascade of microglial activation under pathological conditions.

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.

Expression pattern of galectin-3 in neural tumor cell lines

Galectin‐3 is a member of the galectin family of β‐galactoside‐specific animal lectins. Here we show that galectin‐3 is constitutively expressed in 15 out of 16 glioma cell lines tested, but not by normal or reactive astrocytes, oligodendrocytes, glial O‐2A progenitor cells and the oligodendrocyte precursor cell line Oli‐neu. Galectin‐3 is also expressed by one oligodendroglioma cell line, but not by primitive neuroectodermal tumor and 4 neuroblastoma cell lines tested so far. In all galectin‐3 expressing cell lines, the lectin is predominantly, if not exclusively, localized intracellularly and carries an active carbohydrate recognition domain (shown for C6 rat glioma cells). Moreover, in contrast to primary astrocytes, glioma cells do not or only weakly adhere to substratum‐bound galectin‐3, probably reflecting an unusual glycosylation pattern. Our findings indicate that the expression of galectin‐3 selectively correlates with glial cell transformation in the central nervous system and could thus serve as a marker for glial tumor cell lines and glial tumors. J. Neurosci. Res. 60:45–57, 2000

Chondroitin sulfates expressed on oligodendrocyte-derived tenascin-R are involved in neural cell recognition. Functional implications during CNS development and regeneration

Tenascin‐R (TN‐R), an extracellular matrix constituent of the central nervous system (CNS), has been implicated in a variety of cell–matrix interactions underlying axon growth inhibition/guidance, myelination and neural cell migration during development and regeneration. Although most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN‐R glycoconjugates present on many potential sites for N‐ and O‐glycosylation is presently unknown. Here we provide first evidence that TN‐R derived from whole rat brain or cultured oligodendrocytes expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e., C‐4S and C‐6S, that are recognized by CS‐56, a CS/dermatan sulfate‐specific monoclonal antibody. Based on different in vitro approaches utilizing substrate‐bound glycoprotein, we found that TN‐R‐linked CS GAGs (1) promote oligodendrocyte migration from white matter microexplants and increase the motility of oligodendrocyte lineage cells; (2) similar to soluble CS GAGs, induce the formation of glial scar‐like structures by cultured cerebral astrocytes; and (3) contribute to the antiadhesive properties of TN‐R for neuronal cell adhesion in an F3/F11‐independent manner, but not to neurite outgrowth inhibition, by mechanism(s) sensitive to chondroitinase or CS‐56 treatments. Furthermore, after transection of the postcommissural fornix in adult rat, CS‐bearing TN‐R was found to be stably upregulated at the lesion site. Our findings suggest the functional impact of TN‐R‐linked CS on neural cell adhesion and migration during brain morphogenesis and the contribution of TN‐R to astroglial scar formation (CS‐dependent) and axon growth inhibition (CS‐independent), i.e., suppression of axon regeneration after CNS injury. J. Neurosci. Res. 60:21–36, 2000

Involvement of chondroitin sulfates on brain-derived tenascin-R in carbohydrate-dependent interactions with fibronectin and tenascin-C.

Tenascin-R (TN-R), a matrix glycoprotein of the central nervous system (CNS), has been implicated in a variety of cell-matrix interactions involved in the control of axon growth, myelination and cell adhesion to fibronectin during development and regeneration. While most of the functional analyses have concentrated exclusively on the role of the core protein, the contribution of TN-R glycoconjugates present on many potential sites for N- and O-glycosylation is presently unknown. Here we provide evidence that TN-R derived from adult mouse brain expresses chondroitin sulfate (CS) glycosaminoglycans (GAGs), i.e. C-6S and C-4S, that are recognized by the CS/dermatan sulfate-specific monoclonal antibodies 473 HD and CS-56. Using ligand-binding, cell adhesion and neurite outgrowth assays, we show that TN-R-linked CS GAGs (i) are involved in the interaction with the heparin-binding sites of fibronectin and are responsible for TN-R-mediated inhibition of cell adhesion to a 33/66-kD heparin-binding fibronectin fragment or to FN-C/H I and FN-C/H II peptides, known to participate in fibronectin binding to cell surface proteoglycans; and (ii) partially contribute to the interaction between TN-R and TN-C which, however, does not lead to an interference with TN-R- and TN-C-mediated inhibition of neurite outgrowth when the two molecules are offered as a mixed substrate in culture. Our findings suggest the functional implication of TN-R-linked CS GAGs in matrix interactions with fibronectin and TN-C that are likely to contribute to a modulation of cellular behavior and the macromolecular organization of matrix components in the developing or injured adult CNS.