Astrocyte-derived tissue Transglutaminase affects fibronectin deposition, but not aggregation, during cuprizone-induced demyelination

Astrogliosis as seen in Multiple Sclerosis (MS) develops into astroglial scarring, which is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits axon outgrowth and (re) myelination in brain lesions. This is possibly an important cause for incomplete remyelination in the CNS of early stage MS patients and for failure in remyelination when the disease progresses. In this study we address whether under demyelinating conditions in vivo, tissue Transglutaminase (TG2), a Ca2+-dependent enzyme that catalyses posttranslational modification of proteins, contributes to extracellular matrix (ECM) deposition and/or aggregation. We used the cuprizone model for de- and remyelination. TG2 immunoreactivity and enzymatic activity time-dependently appeared in astrocytes and ECM, respectively, in the corpus callosum of cuprizone-treated mice. Enhanced presence of soluble monomeric and multimeric fibronectin was detected during demyelination, and fibronectin immunoreactivity was slightly decreased in cuprizone-treated TG2(-/-) mice. In vitro TG2 overexpression in astrocytes coincided with more, while knock-down of TG2 with less fibronectin production. TG2 contributes, at least partly, to fibronectin production, and may play a role in fibronectin deposition during cuprizone-induced demyelination. Our observations are of interest in understanding the functional implications of TG2 during astrogliosis

Tissue transglutaminase in astrocytes is enhanced by inflammatory mediators and is involved in the formation of fibronectin fibril-like structures

Abstract Background During multiple sclerosis (MS) lesion formation, inflammatory mediators are produced by microglial cells and invading leukocytes. Subsequently, hypertrophic astrocytes fill the lesion and produce extracellular matrix (ECM) proteins that together form the astroglial scar. This is beneficial because it seals off the site of central nervous system (CNS) damage. However, astroglial scarring also forms an obstacle that inhibits remyelination of brain lesions. This is possibly an important cause for incomplete remyelination of the CNS in early stage MS patients and for failure of remyelination when the disease progresses. Tissue transglutaminase (TG2), a Ca2+-dependent enzyme that can cross-link proteins, appears in astrocytes in inflammatory MS lesions and may contribute to the rearrangement of ECM protein deposition and aggregation. Methods The effect of different inflammatory mediators on TG2 and fibronectin, an ECM protein, protein levels was examined in primary rat microglia and astrocytes by western blotting. Also, TG2 activity was analyzed in primary rat astrocytes by a TG activity assay. To determine the role of TG2 in the deposition and cross-linking of fibronectin, a TG2 inhibitor and TG2 knockdown astrocytes were used. Results Our data show that under inflammatory conditions in vitro, TG2 production is enhanced in astrocytes and microglia. We observed that in particular, astrocytes produce fibronectin that can be cross-linked and aggregated by exogenous TG2. Moreover, inflammatory stimulus-induced endogenously produced TG2 is involved in the appearance of morphological fibril-like fibronectin deposits but does not lead to cross-linked fibronectin aggregates. Conclusions Our in vitro observations suggest that during MS lesion formation, when inflammatory mediators are produced, astrocyte-derived TG2 may contribute to ECM rearrangement, and subsequent astroglial scarring

Tissue transglutaminase in Marmoset experimental multiple sclerosis:Discrepancy between white and grey matter

Infiltration of leukocytes is a major pathological event in white matter lesion formation in the brain of multiple sclerosis (MS) patients. In grey matter lesions, less infiltration of these cells occur, but microglial activation is present. Thus far, the interaction of β-integrins with extracellular matrix proteins, e.g. fibronectin, is considered to be of importance for the influx of immune cells. Recent in vitro studies indicate a possible role for the enzyme tissue Transglutaminase (TG2) in mediating cell adhesion and migration. In the present study we questioned whether TG2 is present in white and grey matter lesions observed in the marmoset model for MS. To this end, immunohistochemical studies were performed. We observed that TG2, expressed by infiltrating monocytes in white matter lesions co-expressed β1-integrin and is located in close apposition to deposited fibronectin. These data suggest an important role for TG2 in the adhesion and migration of infiltrating monocytes during white matter lesion formation. Moreover, in grey matter lesions, TG2 is mainly present in microglial cells together with some β1-integrin, whereas fibronectin is absent in these lesions. These data imply an alternative role for microglial-derived TG2 in grey matter lesions, e.g. cell proliferation. Further research should clarify the functional role of TG2 in monocytes or microglial cells in MS lesion formation

Tissue Transglutaminase Promotes Early Differentiation of Oligodendrocyte Progenitor Cells

Demyelinated lesions of the central nervous system are characteristic for multiple sclerosis (MS). Remyelination is not very effective, particular at later stages of the disease, which results in a chronic neurodegenerative character with worsening of symptoms. Previously, we have shown that the enzyme Tissue Transglutaminase (TG2) is downregulated upon differentiation of oligodendrocyte progenitor cells (OPCs) into myelin-forming oligodendrocytes and that TG2 knock-out mice lag behind in remyelination after cuprizone-induced demyelination. Here, we examined whether astrocytic or oligodendroglial TG2 affects OPCs in a cell-specific manner to modulate their differentiation, and therefore myelination. Our findings indicate that human TG2-expressing astrocytes did not modulate OPC differentiation and myelination. In contrast, persistent TG2 expression upon OPC maturation or exogenously added recombinant TG2 accelerated OPC differentiation and myelin membrane formation. Continuous exposure of recombinant TG2 to OPCs at different consecutive developmental stages, however, decreased OPC differentiation and myelin membrane formation, while it enhanced myelination in dorsal root ganglion neuron-OPC co-cultures. In MS lesions, TG2 is absent in OPCs, while human OPCs show TG2 immunoreactivity during brain development. Exposure to the MS-relevant pro-inflammatory cytokine IFN-gamma increased TG2 expression in OPCs and prolonged expression of endogenous TG2 upon differentiation. However, despite the increased TG2 levels, OPC maturation was not accelerated, indicating that TG2-mediated OPC differentiation may be counteracted by other pathways. Together, our data show that TG2, either endogenously expressed, or exogenously supplied to OPCs, accelerates early OPC differentiation. A better understanding of the role of TG2 in the OPC differentiation process during MS is of therapeutic interest to overcome remyelination failure

Characterization of marmoset EAE lesions and TG2 immunoreactivity.

<p>The normal appearing white matter (NAWM) shows an intact LFB myelin staining (A) and few MRP14 positive macrophages (G). Early active (EA) lesions display myelin degradation (B) and foamy macrophages (H). Late active (LA) lesions show degradation of myelin (C) combined with less MRP14 positive macrophages (I). Inactive (IA) lesions are characterized by an absence of both myelin staining (D) and MRP14 positive macrophages (J). The normal appearing grey matter (NAGM) shows intact myelin fibers (E) and very few MRP14 positive macrophages (K). Cortical grey matter lesions (cGML) show an absence of myelin fibers (F) and presence of MRP14 positive microglia (L). TG2 immunoreactivity is present in endothelium of the vessel walls in NAWM (M). Early active and late active lesions display additional TG2 positive cells (N and O respectively). Inactive lesions show less additional TG2 immunoreactivity (P). Cortical grey matter lesions also show additional TG2 positive cells (R) compared to the endothelial staining in normal appearing grey matter (Q). Scale bar is 20 µm.</p

Combinations of primary and secondary antibodies used for immunoreactive labeling.

<p>FN: fibronectin.</p

β₁-integrin and fibronectin immunoreactivity show co-presence with TG2 positive cells in marmoset EAE lesions.

<p>β₁-integrin (A) and fibronectin (FN) (F) immunoreactivity (green) is found in normal appearing white matter (NAWM) near TG2 (red) in the endothelium of the vessel walls. β₁-integrin (green; B, C, C′, D) appears in early (EA), late (LA) active lesions and inactive (IA) white matter lesions on the cell surface of a subset of TG2 (red) positive cells. β₁-integrin also shows some co-localization with TG2 positive cells in cortical grey matter lesions (cGML) (E, E′). Arrows represent TG2/β₁-integrin double labeled cells. FN (green; G, H, H′, I) appears clearly in the extracellular matrix but also shows co-labeling with a subset of TG2 (red) positive cells in early and late active lesions. Hardly any FN immunoreactivity is present in grey matter lesions (J). Arrows represent TG2/FN double labeled cells. Scale bar is 20 µm. Inserts in figures C′, E′ and H′ represent higher magnifications in which the close association of TG2 positive cells with β₁-integrin or FN (C′, E′ or H′, respectively) can be appreciated. Scale bars in the inserts are 10 µm.</p

Lesion types per animal.

<p>MOG: myelin oligodendrocyte glycoprotein, EA: early active, LA: late active, IA: inactive, cGML: cortical grey matter lesion.</p

Secondary antibodies used.

<p>Secondary antibodies used.</p

TG2 positive cells show co-labeling with Iba-1 in marmoset EAE lesions.

<p>Early (EA) (A-C) and late active (LA) (D-F) lesions show mostly cytoplasmic cellular localization of immunoreactive TG2 (A, D), and cells with membrane labeled Iba-1 (B, E). TG2 positive cells co-label with Iba-1 positive cells (C, F). Inactive (IA) (G-I) lesions show less TG2 positive cells and co-labeling with Iba-1 positive cells seems less apparent (I) Cortical grey matter lesions (cGML) (J-L) show TG2 positive cells co-labeling with Iba-1 positive cells that have radially projecting processes (L), instead of the more rounded morphology seen in white matter lesions (C, F). Arrows represent monocyte-like cells that are either single (top 2 rows) or double labeled (merge), arrowheads represent microglial cells that are either single (top 2 rows) or double labeled (merge). Scale bar is 20 µm.</p