Aggregating proteoglycans (PG) bearing chondroitin sulfate (CS) side chains associate with hyaluronan and various secreted proteins to form a complex of extracellular matrix (ECM) that inhibits neural plasticity in the central nervous system (CNS). Chondroitinase treatment depletes PGs of their CS side chains and enhances neurite extension. Increasing evidence from in vivo models indicates that proteolytic cleavage of the PG core protein by members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of glutamyl-endopeptidases also promotes neural plasticity. The purpose of this study was to determine whether proteolytic action of the ADAMTSs influences neurite outgrowth in cultured neurons. Transfection of primary rat neurons with ADAMTS4 cDNA induced longer neurites, whether the neurons were grown on a monolayer of astrocytes that secrete inhibitory PGs or on laminin/poly-l-lysine substrate alone. Similar results were found when neurons were transfected with a construct encoding a proteolytically inactive, point mutant of ADAMTS4. Addition of recombinant ADAMTS4 or ADAMTS5 protein to immature neuronal cultures also enhanced neurite extension in a dose-dependent manner, an effect demonstrated to be dependent on the activation of MAP ERK1/2 kinase. These results suggest that ADAMTS4 enhances neurite outgrowth via a mechanism that does not require proteolysis but is dependent on activation of the MAP kinase cascade. Thus a model to illustrate multimodal ADAMTS activity would entail proteolysis of CS-bearing PGs to create a loosened matrix environment more favorable for neurite outgrowth, and enhanced neurite outgrowth directly stimulated by ADAMTS signaling at the cell surface.

BackgroundProteoglycan (PG) in the extracellular matrix (ECM) of the central nervous system (CNS) may act as a barrier for neurite elongation in a growth tract, and regulate other characteristics collectively defined as structural neural plasticity. Proteolytic cleavage of PGs appears to alter the environment to one favoring plasticity and growth. Brevican belongs to the lectican family of aggregating, chondroitin sulfate (CS)-bearing PGs, and it modulates neurite outgrowth and synaptogenesis. Several ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are glutamyl-endopeptidases that proteolytically cleave brevican. The purpose of this study was to localize regions of adult CNS that contain a proteolytic-derived fragment of brevican which bears the ADAMTS-cleaved neoepitope sequence. These regions were compared to areas of Wisteria floribunda agglutin (WFA) reactivity, a common reagent used to detect "perineuronal nets" (PNNs) of intact matrix and a marker which is thought to label regions of relative neural stability.ResultsWFA reactivity was found primarily as PNNs, whereas brevican and the ADAMTS-cleaved fragment of brevican were more broadly distributed in neuropil, and in particular regions localized to PNNs. One example is hippocampus where the ADAMTS-cleaved brevican fragment is found surrounding pyramidal neurons, in neuropil of stratum oriens/radiatum and the lacunosum moleculare. The fragment was less abundant in the molecular layer of the dentate gyrus. Mostly PNNs of scattered interneurons along the pyramidal layer were identified by WFA. In lateral thalamus, the reticular thalamic nucleus stained abundantly with WFA whereas ventral posterior nuclei were markedly immunopositive for ADAMTS-cleaved brevican. Using Western blotting techniques, no common species were reactive for brevican and WFA.ConclusionIn general, a marked discordance was observed in the regional localization between WFA and brevican or the ADAMTS-derived N-terminal fragment of brevican. Functionally, this difference may correspond to regions with varied prevalence for neural stability/plasticity.

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...