Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain

doi:10.1016/0014-4827(91)90390-G

Experimental Cell Research

Volume 195, Issue 2, August 1991, Pages 401-411

https://doi.org/10.1016/0014-4827(91)90390-G Get rights and content

Abstract

We have devised a technique that enables one to localize hyaluronate in cultured cells. Cells were probed with the glial hyaluronate binding protein (GHAP) which was itself then visualized by conventional indirect immunofluorescence. The hyaluronate binding properties of this protein have been established. This technique was applied to the study of hyaluronate synthesis in glial cells. These cells do not themselves produce GHAP. O-2A progenitor cells were obtained from the cerebral hemispheres of newborn rats. These cells are bipotential in that they are able to differentiate into either oligodendrocytes or type 2 astrocytes depending on the composition of the culture medium. In cultures of O-2A progenitor cells maintained in the absence of serum, in which large numbers of oligodendrocytes appeared, very little hyaluronate was produced. The galC⁺ cells were invariably hyaluronate negative. Cultures of the same cells, maintained in the presence of 10% FCS, contained large numbers of hyaluronate producing cells. The hyaluronate producing cells were typically small, process-bearing, and GFAP⁺. Some, but not all, were A2B5⁺ and could, therefore, be identified as type 2 (GFAP⁺, A2B5⁺) astrocytes. Type 1 (GFAP⁺, A2B5⁻) astrocytes were also active in the synthesis of hyaluronate, to the extent that they were able to coat their substrate with hyaluronate. Among cells of the O-2A lineage, then, hyaluronate production would appear to be restricted to astrocytes. This may have some bearing on the origin of hyaluronate in the extracellular matrix of CNS white matter.

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HA is retained on the cell surface by different mechanisms: binding to HA receptors (such as transmembrane CD44), interacting with HA binding proteins (such as versican), and anchored via its synthetic enzymes HASs, while being synthesized (Giamanco et al., 2010; Kwok et al., 2010). HA has also been shown to specifically interact with different growth factors and cytokines in the ECM, including TGF-β, bone morphogenetic protein superfamily members, interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNFα), epidermal growth factor (EGF), keratinocyte growth factor, and many other factors (Asher and Bignami, 1991; Spicer and Tien, 2004). In the developing CNS, HA is produced by cells in the neural tube and notochord (Hay and Meier, 1974).
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In a model of astrogliosis in vitro, cultured cortical astrocytes were triggered into a functionally reactive state by an immobilized fragment of the β-amyloid peptide. Induced astrocytes produced an extracellular matrix that inhibited the outgrowth of embryonic CNS axons. Within the extracellular matrix deposited by reactive astrocytes, we found an overall increase in the deposition of chondroitin sulphate that accounted for the inhibition. Specifically, we have detected an increased biosynthesis of a small chondroitin/heparan sulphate proteoglycan that is a potent inhibitor of axon outgrowth. We further suggest that this proteoglycan, or related molecules yet to be discovered, may play a role in gliosis-mediated regenerative failure of CNS axons. Copyright © 1996 ISDN.
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One century ago, Camillo Golgi described ‘perineuronal nets’ enwrapping the cell bodies and proximal dendrites of certain neurons in the adult mammalian central nervous system and suggested that they represent a supportive and protective scaffolding. Although other neuroanatomists validated the existence of these nets on selected neurons in the adult brain, there was a lack of agreement on their origins, composition and function. The application of modern molecular and ultrastructural methods has brought new insights and a renewed interest in these classic observations. Recent data suggest that perineuronal nets result from the visualization of extracellular matrix molecules that are confined to the space interposed between glial processes and the nerve cells that they outline. The material confined to these spaces can be visualized selectively by antibodies directed to glycoproteins (e.g., tenascin and restrictin/janusin), proteoglycans (e.g., chondroitin sulfates), markers for hyaluronan as well as by lectins recognizing N-acetylgalactosamine and by monoclonal antibodies directed to epitopes on unknown molecules (e.g., HNK-1, VC1.1 and Cat 301). This review examines the emerging clarification of classical observations of perineuronal nets and the functional implications suggested by their molecular composition. Also discussed are studies that further extend observations on the time of development and of the specificity in the occurrence of perineuronal nets. In the adult brain the molecules constituing the ‘perineuronal nets of matrix’ could serve as recognition molecules between certain neurons and their surrounding cells and participate in the selection and consolidation of their relationship.
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Hyaluronan was localized in postimplantation mouse embryos using CD44, the principal hyaluronan receptor. The specificity of CD44 receptor-globulin labelling was confirmed using Streptomyces hyaluronidase, anti-chondroitin sulfate antibody, and other receptor globulins. Our major findings are summarized as follows
1. Implantation of the blastocyst into the uterine wall triggers a rapid loss of hyaluronan from the extracellular matrix of decidual cells on the anti-mesometrial side of the uterus.
2. Hyaluronan appears early in development in the yolk cavity, and the basement membranes of primitive ectoderm and primitive endoderm.
3. During gastrulation, mesodermal cells enter a hyaluronan-rich environment, but lack a pericellular hyaluronan coat themselves.
4. In limb bud embryos, hyaluronan is present throughout the cranial mesenchyme, but is generally not present in the branchial bars, somites, or limb buds.
5. At mid-gestation, hyaluronan is present in the axial skeleton, craniofacial mesenchyme, endocardial cushions of the heart, smooth muscle of the gastrointestinal tract, and connective tissue throughout the body.
The pattern of hyaluronan expression in the day 13 fetus is nearly identical to the published distribution of transforming growth factor β (TGF β), suggesting a close functional relationship between these molecules. Together, the results suggest that hyaluronan is involved in the formation of early mesoderm, differentiation of craniofacial mesenchyme, and morphogenesis of the axial skeleton.
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^☆: This work was supported by NIH Grant NS 13034 and by the Department of Veterans Affairs.

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Localization of hyaluronate in primary glial cell cultures derived from newborn rat brain☆

Abstract

Int. J. Dev. Neurosci

J. Biol. Chem

J. Immunol. Methods

Arch. Biochem. Biophys

J. Biol. Chem

J. Biol. Chem

Exp. Cell Res

Exp. Eye Res

Exp. Neurol

Brain Res

Biochem. Biophys. Res. Comm

Physiol. Rev

J. Neurochem

Biochemistry

J. Neurochem

J. Neurochem

J. Neurochem

J. Neurosci. Res