Altered production and proteolytic processing of brevican by transforming growth factor β in cultured astrocytes

Abstract

Brevican, a proteoglycan of the lectican family, inhibits neurite outgrowth and may also stabilize synapses. Little is known about its expression or function in vitro. This study seeks to determine whether a brevican-containing matrix is present in neural cultures, and if so, how the production of brevican may be modulated. To accomplish this, the content of brevican and its proteolytic fragments were measured in primary cultures of neurons, astrocytes and microglia after treatment with cytokines. These experiments revealed that astrocytes and neurons express several isoforms of brevican, whereas microglia do not produce this proteoglycan. Cleavage fragments of brevican were found primarily in neuronal and astrocyte culture medium. ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs), a protease that selectively cleaves lecticans, was detected in cultures of neurons, astrocytes and microglia. When astrocytes were challenged with various cytokines, it was found that treatment with transforming growth factor b (TGFb) resulted in a marked increase in intact brevican in the culture medium that was accompanied by a trend for a decrease in ADAMTS-generated fragments of brevican and apparent ADAMTS activity. Thus, TGFb may play a role in neuronal plasticity through its regulation of brevican and the activity of the ADAMTSs. Keywords: ADAMTS4, extracellular matrix, glial cell, neuron, protease, proteoglycan. In the nervous system, extracellular matrix (ECM) exists in the neuropil and in discrete reticular networks around the surface of neurons, termed perineuronal nets. The matrix is thought to exist as a complex of aggregated molecules whose components include hyaluronan, tenascin-R and one or more members of the lectican family of proteoglycans (PGs). Each of the PGs in the lectican family, brevican, versican, aggrecan and neurocan, are variably substituted with chondroitin sulfate (CS) chains in the central domain of the core protein, and the globular N-terminus binds hyaluronan, whereas the C-terminus binds tenascin-R (Yamaguchi 2000). Among the lecticans, brevican is the most abundant PG in the ECM of the CNS, and is secreted by astrocytes and neurons (Seidenbecher et al. 1998;Jaworski et al. 1999) where it is located peri-synaptically (Hagihara et al. 1999) in association with synaptic proteins (Seidenbecher et al. 2002). This aggregated complex of matrix is thought to confer stability in neural networks and inhibit plasticity after injury (Rhodes and Fawcett 2004). Interestingly, treatment of the tissue with chondroitinase markedly improves plasticity, including neurite outgrowth, after injury in vivo (Moon et al. 2001).In adult CNS, secreted brevican is found as a CS-substituted, high molecular weight isoform (Fig. 1a), a 145 kDa unsubstituted core protein (Fig. 1b), and as 55 (Fig. 1c) and 80 kDa (Figs 1d and e) N-and C-terminal fragments as a result of proteolytic cleavage (Yamada et al. 1994). The major family of proteases responsible for the generation of these lower molecular wei...

Total number, distribution, and phenotype of cells expressing chondroitin sulfate proteoglycans in the normal human amygdala

“…The lectin wisteria floribunda agglutinin, as well as other plant-derived lectins, binds to the N-acetyl-galactosamine component of the chondroitin sulfate chains and represents a useful tool to detect CSPGs in brain tissue (Bruckner, et al, 1993, Celio, et al, 1998, Hartig, et al, 1992, Nakagawa, et al, 1986, Pantazopoulos, et al, 2006. In adult rodents, CSPGs secreted in the ECM, and associated with perineuronal nets, are synthesized and expressed by several different cell types, including neurons, astrocytes, oligodendrocytes and NG2-expressing oligodendrocyte precursor cells , Hamel, et al, 2005, John, et al, 2006, McKeon, et al, 1999, Thon, et al, 2000, Viapiano, et al, 2006.…”

“…The lectin wisteria floribunda agglutinin, as well as other plant-derived lectins, binds to the N-acetyl-galactosamine component of the chondroitin sulfate chains and represents a useful tool to detect CSPGs in brain tissue (Bruckner, et al, 1993, Celio, et al, 1998, Hartig, et al, 1992, Nakagawa, et al, 1986, Pantazopoulos, et al, 2006. In adult rodents, CSPGs secreted in the ECM, and associated with perineuronal nets, are synthesized and expressed by several different cell types, including neurons, astrocytes, oligodendrocytes and NG2-expressing oligodendrocyte precursor cells , Hamel, et al, 2005, John, et al, 2006, McKeon, et al, 1999, Thon, et al, 2000, Viapiano, et al, 2006.Emerging evidence points to a role for CSPGs in the pathophysiology of several neurological disorders, including Alzheimer's disease, multiple sclerosis and temporal lobe epilepsy (Perosa, et al, 2002, Sobel, et al, 2001, Viapiano, et al, 2006. Their role in neural injuries has also been extensively investigated, as some CSPGs inhibit neurite regeneration following neural injuries (McKeon, et al, 1991, Morgenstern, et al, 2002, Viapiano, et al, 2006.…”

Modulation of Neuritogenesis by Astrocyte Muscarinic Receptors

“…Astroglial cells express in their membranes or release molecules such as laminin-1 (11), N-cadherin (12,13), neural cell adhesion molecule (12), and fibronectin (14), which can promote neurite outgrowth during development and during regeneration after a lesion (15). They also release neurite-inhibiting factors such as the chondroitin sulfate proteoglycans neurocan (16) and brevican (17,18). Inhibitory proteoglycans released by glial cells during development contribute to the establishment of the correct architecture by preventing neurite growth in every direction (19) and, after brain lesion, contribute to the nonpermissive environment, preventing regeneration of axons (16).…”

Real-Time PCR and Immunocytochemical Study of Chondroitin Sulfate Proteoglycans after Scratch Wounding in Cultured Astrocytes / PCR I IMUNOCITOHEMIJSKA STUDIJA EKSPRESIJE HONDROITIN-SULFATNIH PROTEOGLIKANA NAKON POVREDE ASTROCITA U KULTURI

“…Although it has been confirmed both in vivo and in vitro that astrocytes are the major source of brevican (35)(36)(37), results of qRT-PCR analysis from our cultures suggested that brevican transcript was drastically less present compared to neurocan and phosphacan. It is important to note that SW induced a decrease in expression to about half of the value seen in control.…”