The role of proteoglycans in regeneration and plasticity

Chondroitin sulfate proteoglycans (CSPGs) are up-regulated in glial scar tissue and inhibit axon regeneration. Not only are the protein cores up-regulated, but also there is more glycosaminoglycan (GAG) attached to them. The final stage of GAG synthesis is sulfation, which can occur in three positions. Both 6-sulfated GAG and the sulfotransferase that sulfates N -acetylgalactosamine in the 6 position is specifically up-regulated in glial scar tissue, in inhibitory glial cells and in astrocytes treated with tumour growth factor α (TGF-α) and TGF-β. Removal of GAG chains by digestion with chondroitinase or inhibition of GAG synthesis with chlorate or β-D-xylosides, removes much of the inhibition from CSPGs in vitro . We therefore tested to see whether GAG digestion by chondroitinase would promote axon regeneration in vivo . We first treated mechanical lesions of the nigrostriatal tract and saw regeneration of about 4% of axons back to their target. Next, dorsal column lesions of the spinal cord at C4 were treated. Both sensory and corticospinal axons regenerated in treated cords, and there was rapid return of function in beam and grid walking tests. The return of function was so rapid that we hypothesized that some of it might be due to enhanced plasticity. Many neuronal cell bodies and dendrites are coated in thick perineuronal nets of inhibitory CSPGs and tenascin-R, which would certainly be expected to prevent the formation of new synapses. We therefore tested the effects of chondroitinase treatment in a plasticity model: ocular dominance shift in the visual cortex following monocular deprivation. Monocular deprivation in adult animals normally produces no ocular dominance shift. However, in adult animals in which the cortex was treated with chondroitinase, there was a large shift in response to monocular deprivation.     

Many cells in rat bone marrow have cell-surface Thy-1 antigen.

Thy-1.1 and Thy-1 xenoantigenic determinants were detected at the cell surface of many rat bone marrow cells. The absorptive capacity of bone marrow cells was 6-10% of that of thymocytes for Thy-1 antigenic determinants, and 30-45% of rat bone marrow cells were specifically labeled with anti-Thy-1 antibody as detected by autoradiography. Thus, while mice and rats are similar in having large amounts of Thy-1 in brain and thymocytes, they differ in that the rat lacks the antigen in most peripheral T cells and expresses it in a large number of bone marrow cells; the opposite is true in the mouse.     

A role for cAMP in regeneration of the adult mammalian CNS

Injury to the adult mammalian central nervous system (CNS) often results in permanent loss of sensory and motor function. This is due to the failure of injured axons to regenerate. The inhibitory nature of the CNS can be attributed to several factors, including formation of the glial scar, the presence of several molecules, associated with myelin, which inhibit axonal regrowth, and the intrinsic growth state of these neurons. Encouraging regeneration in the adult mammalian CNS therefore will require targeting one or all of these factors following injury. Here we illustrate recent work from our laboratory that identifies some of the signalling components involved in modulation of the intrinsic growth state of adult neurons. When activated, these signalling pathways can induce axonal regeneration in the presence of the myelin-associated inhibitors both in vitro and in vivo.     

Thy-1-mediated activation of rat mast cells: the role of Thy-1 membrane microdomains.

The glycosyl-phosphatidylinositol (GPI)-anchored glycoprotein Thy-1 is one of the most abundant molecules expressed on the surface of rat mast cells and rat basophilic leukemia (RBL) cells. The finding that Thy-1 from detergent-solubilized RBL-2H3 cells forms complexes with src-related protein-tyrosine kinase p56/p53lyn suggested that this kinase may play a key role in Thy-1-mediated mast-cell activation. The molecular mechanism of this activation is, however, unknown. Here we show that in RBL-2H3-derived cells extracted by the standard procedure with several non-ionic detergents, the majority of Thy-1 and p56/p53lyn were not released into postnuclear supernatant but remained associated with the detergent-resistant cytoskeletal/nuclear fraction. Pretreatment of the cells with the cholesterol-complexing agents, saponin or digitonin, resulted in complete solubilization of Thy-1 and p56/p53lyn in non-ionic detergents and dissociation of the complexes; this implies that cholesterol plays a crucial role in stabilization of the complexes. This conclusion was supported by double immunofluorescence colocalization experiments which also allowed us to estimate the size of the insoluble complexes to be about 0.1 micron. Sequential treatment with saponin and Nonidet P-40 was used to fractionate tyrosine-phosphorylated proteins during Thy-1-mediated activation of RBL-2H3 cells. Among the soluble cytoplasmic proteins the most dramatic change in tyrosine phosphorylation was found in pp72, whereas pp40 and pp33 were found mainly in the membrane fraction. Our data suggest that surface aggregation of GPI-anchored Thy-1 molecules leads to aggregation of p56/p53lyn kinase located in the same membrane microdomain, followed by transphosphorylation of both soluble and membrane-bound substrates.     

Analysis in deoxycholate of three antigenic specificities associated with the rat Thy-1 molecule.

Three antigens similar in tissue distribution can be identified on rat thymocytes; the Thy-1.1 antigen, a rat specific xenoantigen, and a rat-mouse cross-reacting xenoantigen. To determine if these three antigens were on the same molecule their behavior in detergent-solubilized extracts from thymocytes was studied. Membrane fragments containing Thy-1.1 activity were prepared by a rapid method involving the use of Tween-40 detergent, and were solubilized in deoxycholate. The 150 000 x g supernatant from this extract contained approximately 50% of the original Thy-1.1 and xenoantigen activity. The supernatant was chromatographed on Sephadex G-200, and subjected to zone sedimentation on sucrose gradients in H2O and 2H2O to determine the hydrodynamic properties of the antigens. The three antigens migrated in identical fashion in all cases, and behaved as a molecule of 28 000 daltons molecular weight. When the antigenically active fraction, recovered after chromatography on Sephadex G-200, was passed through an immunoabsorbent consisting of rabbit antibody to one of the xenoantigens, all three antigens were equally depleted compared with passage through a control column. The results of these experiments suggested that Thy-1.1 antigen and the two xenoantigens were closely associated and most probably all on the Thy-1 molecule.     

Axonal regeneration in the mammalian CNS

Axons in the adult central nervous system (CNS) of higher vertebrates are in general not capable of regeneration after injury. This is in contrast to the situation in lower vertebrates (fish and in part amphibia) and the mammalian peripheral nervous system (PNS), where severed axons can regenerate, correct synaptic connections can be formed again, and function can be restored. This enigma has been the subject of extensive studies in the last decades and a large amount of data has been accumulated. This article reviews recent developments in experimental approaches to axonal regrowth in the mammalian CNS focusing mostly on in-vivo systems.     

Genetic evidence for the role of Thy-1 in neurite outgrowth in the mouse

Non-neuronal accessory cells of mouse dorsal root ganglion cultures secrete a complex that stimulates neurite outgrowth in neonatal sympathetic ganglion neurons. A monoclonal antibody that binds to these two cell types also immunoprecipitates neurite outgrowth complex from mouse conditioned medium and binds to mouse brain tissue. Genetic analysis of the component recognized by the antibody revealed that it maps to the Thy-1 locus on mouse chromosome 9. Further studies of cell-type specificity, sedimentation analysis and antibody competition, confirmed that it is indistinguishable from the product of the Thy-1 locus. The finding of an association between Thy-1 and neurite outgrowth complex in the mouse argues for a role of the Thy-1 locus in the interactions of neurons with their surroundings.     

Regulation of cytokine production in the bone marrow during postcytostatic regeneration by SC-1+ and Thy-1+ cells

The role of SC-1⁺ and Thy-1⁺ cells in the regulation of interleukin-3 production and erythropoietic and colony-stimulating activity of bone marrow cells is studied in cyclophosphamide-treated mice. It is shown that elimination of SC-1⁺ and Thy-1⁺ cells has no effect on the production of these cytokines during the early postcytostatic period and upregulates production of some factors stimulating proliferation and colony-formation in the spleen. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 4, pp. 374–377, April, 1998     

Regulation of lymphocyte activation by the cell-surface molecule CD22

Accessory molecules play an important role in the regulation of lymphocyte activation mediated by the B-cell antigen receptor (BCR). CD22 is one such accessory molecule expressed on B-lineage cells. Here, Che-Leung Law and colleagues review current knowledge on the structure-function relationship between CD22 and the BCR, discuss the role of CD22 as a cell-adhesion molecule and suggest models for potential in vivo functions of CD22.     

Thy-1 molecule associates with protein tyrosine kinase(s) in rat mesangial cells

Glycophosphatidylinositol (GPI)-linked Thy-1 molecules, well known cell surface markers of murine T cells, are present on the glomerular mesangial cells of the rat kidney. The administration of anti-Thy-1.1 MoAbs 1-22-3 and OX-7 to rats induces severe and mild complement-dependent mesangial proliferative glomerulonephritis, respectively. In order to determine whether protein-tyrosine kinase (PTK) activity is associated with Thy-1 molecules on rat mesangial cell surface, we performed an immune complex kinase assay, using anti-Thy-1 MoAbs 1-22-3 and OX-7, followed by reimmunoprecipitation with anti-phosphotyrosine, anti-fyn, anti-lck and anti-lyn antibodies. Physical association of PTK, p59^fyn and p56/53^lyn with Thy-1 molecules was demonstated in cultured rat mesangial cells. The activities of these kinases detected in MoAb 1-22-3 precipitates were higher than those in MoAb OX-7 precipitates. These results suggest that Thy-1 molecule transduces some signals also in rat mesangial cells.     

Thy-1及Thy-1mRNA在肺纤维化大鼠肺组织中的表达及相关性研究

目的： 以博莱霉素（BLM）致肺纤维化大鼠为动物模型, 研究胸腺细胞分化抗原-1（thymocyte differentiation antigen 1,Thy-1/CD90）及Thy-1mRNA的表达,分析Thy-1及Thy-1mRNA与肺纤维化形成之间的关系,为研究肺纤维化的发病机制提供新的思路。方法: 将大鼠随机分为实验组及对照组,用BLM气管注入法建立动物模型;分别留取第1 d、3 d、7 d、14 d、28 d肺组织, 测定羟脯胺酸(HYP)、Thy-1 、Thy-1mRNA的表达情况。结果: 正常肺间质中成纤维细胞多数表达Thy-1,阳性率为88.01%+4.12%;随着纤维化程度加重,Thy-1表达呈进行性减少;Thy-1表达阳性率与HYP之间呈负相关;Thy-1mRNA在肺纤维化形成过程中无明显变化,与Thy-1表达之间无相关性。结论: 在肺纤维化形成过程中存在成纤维细胞表达Thy-1减少,Thy-1细胞可能是胶原的主要来源细胞;Thy-1的减少与Thy-1mRNA无关。     

Thy-1 differentiation protein and monocyte-derived cells during regeneration and aging of human placental villi.

PROBLEM: The classification of placental villi was reviewed, and regeneration of villous trees in mature human placentae was examined. METHOD OF STUDY: Expression of Thy-1 by placental fibroblasts and pericytes, and markers of endothelial cells and monocyte-derived cells were studied by immunohistochemistry and image analysis. RESULTS: Villous regeneration consists of: (i) dedifferentiation of mature ramuli into young stem villi producing mesenchymal villi; (ii) differentiation of mesenchymal villi into immature intermediate villi; and (iii) differentiation of immature intermediate villi into transitory intermediate villi, branching into the precursors of mature intermediate and terminal villi. These processes are associated with dedifferentiation and redifferentiation of placental monocyte-derived cells. Significant changes of Thy-1 expression by fibroblasts and pericytes accompany aging and degeneration, as well as regeneration of placental villi. CONCLUSIONS: Villous aging and degeneration in normal mature human placenta is compensated by regeneration of villous trees. Lack of villous regeneration may cause chronic fetal distress, due to the increasing demands of the growing fetus on the remaining terminal villi.     

Astrocyte intermediate filaments in CNS pathologies and regeneration

Astroglial cells are the most abundant cells in the mammalian central nervous system (CNS), yet our knowledge about their function in health and disease has been limited. This review focuses on the recent work addressing the function of intermediate filaments in astroglial cells under severe mechanical or osmotic stress, in hypoxia, and in brain and spinal cord injury. Recent data show that when astrocyte intermediate filaments are genetically ablated in mice, reactive gliosis is attenuated and the course of several CNS pathologies is altered, while the signs of CNS regeneration become more prominent. GFAP is the principal astrocyte intermediate filament protein and dominant mutations in the GFAP gene have been shown to lead to Alexander disease, a fatal neurodegenerative condition in humans.     

Evidence that the Thy-1 molecule is the target for T cell mitogenic antibody against brain-associated antigens

Rabbit anti-mouse brain (RaMBr) antiserum can induce Lyt-1⁺, Lyt-2^?, T cells to proliferate and stimulates the same T cell subset to induce B cell proliferation. The aim of this report is to demonstrate that the mitogenic determinant recognized on the T cell surface by RaMBr antiserum is located on the Thy-1 molecule expressing the products of the Thy-1^a and Thy-1^b alleles. Evidence is drawn from serological and genetic experiments. The brain T cell cross-reactive, mitogenic determinant is not expressed on Thy-1^? mutants of the BW5147 T cell lymphoma that fail to express the Thy-1 molecule but do express other T cell surface proteins such as T-200 and gp 69, 71. Monoclonal anti-Thy-1.1 and anti-Thy-1.2 antibodies block the binding to the appropriate T cells of the majority of the serum antibody from RaMBr antiserum. The absorption of mitogenic antibody was blocked in a similar fashion, thus demonstrating the close association of the determinant and the Thy-1 antigen defined by monoclonal alloantibodies. The mitogenic and Thy-1.1 determinants are probably located on the same molecule because of the data obtained with the BW5147 Thy-1^? mutants and the observation that Thy-1^a T cells, which express a lower level of surface Thy-1 than Thy-1^b T cells, also express lower levels of the determinant recognized by RaMBr antiserum. Furthermore, in (AKR × DBA/2)F₁ mice (Thy-1^a/b) which express less Thy-1.1 antigen than Thy-1.2 at the surface, the mitogenic determinant was found to be prefentially associated with Thy-1.2. The coordinated genetic control of the surface levels of the Thy-1 determinant and the mitogenic determinant suggests that both determinants are situated on the same molecule in the T cell membrane.     

Fibroblast subsets in the human orbit: Thy-1+ and Thy-1- subpopulations exhibit distinct phenotypes

An emerging concept is that fibroblasts are not homogeneous, but rather consist of subsets, capable of producing regulatory mediators that control regional inflammatory responses. Fibroblasts are key effector cells in Graves' ophthalmopathy, responsible for the connective tissue remodeling, and are a rich source of inflammatory mediators. The purpose of this research was to characterize subsets of the fibroblasts in the human orbit. The strategy used was to define fibroblast subpopulations based on surface expression of the Thy-1 antigen. Fibroblast strains derived from human orbital connective tissue exhibit heterogeneous Thy-1 expression. We show, for the first time, separation of orbital fibroblasts into functionally distinct Thy-1+ and Thy-1- subsets using magnetic beading techniques. Both subsets produced the pro-inflammatory cytokine interleukin-6 (IL-6) after stimulation with IL-1beta or the CD40 pathway, whereas Thy-1+ fibroblasts produced higher levels of prostaglandin endoperoxide H synthase-2 (PGHS-2) and prostaglandin E2 (PGE(2)). Thy-1- fibroblasts produced more IL-8 than Thy-1+ fibroblasts, and when treated with interferon-gamma (IFN-gamma) up-regulated MHC class II expression more robustly. Furthermore, CD40 was expressed in a bimodal distribution within each fibroblast subset. These observations suggest that fibroblast subsets in the human orbit play distinct roles in the regulation of immune and inflammatory responses crucial in the initiation and development of thyroid-associated ophthalmopathy.     

Thy-1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT

Clustering of the glycosyl-phosphatidylinositol (GPI)-anchored protein Thy-1 on the cell surface leads to T cell activation. However, despite the similarity to TCR-mediated events, cell signaling triggered by Thy-1 crosslinking, reportedly occurs in a manner independent of the TCR/CD3 complex. To investigate the relationship between responses resulting from Thy-1 or TCR engagement, a biochemically well defined system employing only affinity purified antibodies was used to crosslink these surface molecules and activation was assessed by monitoring tyrosine phosphorylation, intracellular calcium influx and IL-2 production. By these criteria, anti-CD3 mAbs moderately activated EL-4 thymoma or 2B4 hybridoma cell lines, while costimulation with anti-Thy-1-mAb strongly enhanced TCR signaling. Furthermore, a Thy-1 loss mutant cell line, did not respond to stimulation through CD3 despite expressing all essential signaling molecules. Together these results emphasized the existence of a poorly appreciated mutual interdependence between Thy-1 and CD3 for efficient cellular signaling. Thy-1/CD3-mediated activation enhanced mostly tyrosine phosphorylation of a 40 kDa protein which was identified as a transmembrane protein lacking N-linked oligosaccharides. These biochemical properties are identical to those described for a recently cloned adaptor protein called 'Linker for Activation of T cells' (LAT). Indeed, polyclonal Abs raised against a LAT-peptide (amino acids 103-131) specifically recognized the 40 kDa protein. LAT is present in microdomains of the plasma membrane enriched in sphingolipids, cholesterol, GPI-anchored proteins and a variety of signaling molecules. By contrast, the TCR/CD3 complex is excluded from these domains at least until stimulation takes place. Hence, we propose that Thy-1 promotes TCR/CD3 dependent signaling by facilitating LAT phosphorylation on tyrosine and the subsequent recruitment of downstream effector molecules.     

Action of fragments of the cocaine molecule on the CNS

The effect of ecgonine, tropine, tropinone and some of their derivatives, tropane, N-methylpyrrolidine, and N-methylpiperidine on impulse summation in the CNS, the conditioned avoidance reflex, antagonism with hexobarbital, synergism with cocaine, and also their toxicity (LD₅₀) were investigated experimentally. Benzoylecgonine, the methyl ester of ecgonine, ecgonine itself, tropine, pseudotropine, carbomethoxytropinone, tropinone, tropane, N-methylpyrrolidine, and N-methylpiperidine were shown to have a definite stimulating effect on the activity of the CNS and, in this respect, are similar to cocaine. It is concluded that compounds whose molecules contain the structure of tropane or one of its fragments have a central stimulant effect and that the stimulant action of cocaine on the CNS may be associated with the tropane moiety of its molecule.Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 10, pp. 435–438, October, 1978.     

DNA polymorphism in the human Thy-1 gene

Thy-1 is a membrane glycoprotein expressed predominantly in brain tissue and occasionally in lymphoid tissue. The human Thy-1 gene is located on chromosome 11q22.3. Although two allelic forms of Thy-1 exist in mice (Thy-1.1 and Thy-1.2), no allelic forms have been described for the human Thy-1 gene. We describe a polymorphic MspI site within the human Thy-1 gene that distinguishes two alleles, 8 and 9, which are represented in a northern European population at frequencies of 0.7 and 0.3, respectively. Thy-1, therefore, provides a potentially useful marker to identify linkages with human disease genes located near 11q22.