Injury to the central nervous system (CNS) is often debilitating and compounded by little hope of recovery owing to the fact that once neural networks in the CNS are severed, they are difficult to re-establish. Predominantly, this is because the properties of myelin-associated proteins and other proteins that compose a glial scar impede the growth of axons toward their target. The glial scar is an accumulation of reactive astrocytes and extracellular matrix (ECM) molecules such as chondroitin sulfate (CS)-substituted PGs, tenascin and other proteins that inhibit the re-growth of axons and the migration of certain cells into the damaged region (Davies, et al., 1999, Davies, et al., 1997, Laywell, et al., 1992). Indeed, the CS side chains of PG molecules are classical inhibitors of neurite outgrowth both in vitro and in vivo (Carulli, et al., 2005, Silver and Miller, 2004, Snow, et al., 2001).
Lecticans is the term for the family of hyaluronic acid-binding PGs that regulate cell adhesion, migration and neurite outgrowth in the CNS and include brevican, aggrecan, neurocan and versican (Handley, et al., 2006). Long unbranched, sulfated, highly negatively-charged CS chains are covalently bound to the central domain of lecticans and discourage growth cone motility and neurite elongation, however, even when these glycosaminoglycan polymers are removed from the core proteins by chondroitinase treatment (Pizzorusso, et al., 2002), significant neurite inhibition is retained by versican (Schmalfeldt, et al., 2000), but not by brevican (Miura, et al., 2001), at least in vitro. The enduring biological action may be inherent to the core PG protein itself or it may result from interactions with other ECM molecules such as hyaluronan or tenascin-R. In vivo, intermolecular interactions among lecticans, hyaluronan and tenascin result in the formation of a mesh-like lattice in the matrix of the CNS that inhibits neural plasticity (). To facilitate plasticity, there should be a means to relieve the inhibition afforded by the PG, however, the absence of an endogenous, extracellular chondroitinase to remove CS chains is a limiting factor. So exploiting a mechanism that occurs in vivo may be a feasible way to re-establish plasticity in the brain. Increased expression and activation of endogenous proteases that cleave the PG core would be one mechanism to enhance neural plasticity, by loosening the association and interaction among the matrix components that inhibit plasticity (Yamaguchi, 2000) ().
Figure 1 Proteoglycan (lectican)-tenascin-hyaluronan matrix complex in the CNS and lectican cleavage by ADAMTSs. (A), Intact complexes of extracellular matrix form an inhibitory boundary toward neurite outgrowth by hyaluronic acid binding to the N-terminus, tandem (more ...)
The ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) are multi-domain, metalloproteinases that have notable roles in angiogenesis, collagen processing, blood coagulation, cell migration, and arthritis and several family members are glutamyl-endopeptidases that cleave lecticans (Porter, et al., 2005). These secreted proteases share similar functional domains, including a pro-protease, metalloproteinase, disintegrin-like, cysteine-rich and spacer domains. Activation of the pro-protease likely occurs by furin-mediated cleavage of the pro-domain at the N-terminus, and further C-terminal truncations are necessary to fully activate the enzyme (Wang, et al., 2004) (Gao, et al., 2004) (Kuno, et al., 1999). The interaction of the ADAMTS domains with their substrates is complex and may involve binding via the thrombospondin type 1 motif and/or sequences in the C-terminal spacer or cysteine-rich region of the molecule (Flannery, et al., 2002, Kashiwagi, et al., 2004, Tortorella, et al., 2000).
ADAMTSs, especially ADAMTS 1, 4, 5, 9 and 15 are expressed in brain and brain pathologies (Cross, et al., 2006, Cross, et al., 2006, Haddock, et al., 2006, Hurskainen, et al., 1999, Jungers, et al., 2005, Yuan, et al., 2002) (our unpublished observations), and each of these proteases is active in cleaving PGs. Several ADAMTSs have been shown to be elevated in human neurodegenerative disease and animal models of brain injury. ADAMTS1, but not ADAMTS5, appears to be up-regulated in Down syndrome, Pick’s disease and Alzheimer’s disease (Miguel, et al., 2005). ADAMTS4 and ADAMTS1 mRNA was markedly elevated in the hippocampus of rats in response to kainate-induced excitotoxic lesion (Yuan, et al., 2002), and ADAMTS1 expression was increased in the spinal cord of rodents having undergone axotomy (Sasaki, et al., 2001), indicating that these proteases may be increased in response to injury or during an inflammatory response.
Anecdotal, but growing evidence indicates that metalloprotease activity is important in mechanisms of neural plasticity. Nerve growth factor treatment of PC-12 cells results in MMP-3 expression (Fillmore, et al., 1992, Machida, et al., 1989), and MMP-3 is essential in promoting PC12 cell growth cone invasiveness through an artificial basal lamina (Nordstrom, et al., 1995). More recently, excitotoxic lesion in the brain was shown to result in the expression of MMP-9 in the hippocampus (Szklarczyk, et al., 2002, Zhang, et al., 1998) and neuritic sprouting observed in the dentate gyrus after a lesion of entorhinal cortex was blocked by administration of a broad spectrum MMP inhibitor (Reeves, et al., 2003). These actions focus toward a role for the matrix-degrading metalloproteinases in neural plasticity. We recently demonstrated that active ADAMTSs which cleave lecticans are elevated in the dentate gyrus terminal zone during the period of neuritic sprouting after entorhinal cortex lesion (Mayer, et al., 2005). Taken together, these studies support the hypothesis that remodeling of ECM may be an important component in processes of neural and synaptic plasticity. The purpose of this study was to directly test the hypothesis that lectican-degrading activity may promote neurite outgrowth over an ECM that contains inhibitory PGs. We grew primary cultured neurons that were either secreting ADAMTS4 via a transfected expression vector or were exposed to ADAMTSs by direct addition of recombinant protein to the media. In some of these experiments, neurons were grown on an astrocyte monolayer that had previously been shown to deposit brevican in the ECM (Hamel, et al., 2005, John, et al., 2006). Unpredictably, our results show that ADAMTS4, and other ADAMTSs promote neurite outgrowth in primary cultured rat neurons via a mechanism that appears to be independent of its proteolytic activity. In addition, intracellular signaling, appropriate for neurite outgrowth, is induced in ADAMTS-treated neurons.