Multimodal signaling by the ADAMTSs (a disintegrin and metalloproteinase with thrombospondin motifs) promotes neurite extension

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Abstract

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.

Introduction

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 towards 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 protein 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). 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 (Fig. 1A). 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) (Fig. 1B).

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., 2004a, Gao et al., 2004b, 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 Flannery et al., 2002, Kashiwagi et al., 2004, Tortorella et al., 2000).

ADAMTSs, especially ADAMTSs 1, 4, 5, 9 and 15 are expressed in brain and brain pathologies (Cross et al., 2006a, Cross et al., 2006b, 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 were 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., 2003). 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.

Section snippets

PG degradation assay

Highly negatively-charged molecules, including PGs, present in whole rat brain extracts were bound to and eluted from a DEAE matrix as described (Yamada et al., 1994). Briefly, rat brain tissue (1 g) was placed in 10 ml, ice cold, 4 mM HEPES at pH 8.0, 0.15 mM NaCl, 0.1% Triton-X-100 containing 2 μM 1,10 phenanthroline (Sigma, St. Louis, MO) and protease inhibitor cocktail (set III, Calbiochem/EMD Biosciences, San Diego). The tissue was disrupted in a teflon–glass homogenizer and the whole

Degradation of purified PG by recombinant ADAMTSs

To ensure that the human recombinant ADAMTS proteases used in these experiments were effective in degrading brevican purified from rat brain, human recombinant ADAMTS1, ADAMTS4 and ADAMTS5 were incubated with DEAE-extracted PGs. Soluble homogenate of rat brain was applied to a DEAE column and highly negatively-charged species were eluted with 1 M NaCl. These proteins were incubated with active or heat-inactivated preparations of the human recombinant ADAMTSs. At the end of a 3 h incubation

Discussion

In vivo studies have provided the most direct evidence to date for the involvement of matrix-degrading metalloproteinases in neural plasticity in the CNS. These demonstrated increased expression of matrix-cleaving proteinases during conditions of regeneration and/or neuronal sprouting (Mayer et al., 2005, Szklarczyk et al., 2002, Yuan et al., 2002) or synaptogenesis (Kim et al., 2005) and that blockade of metalloproteinase activity during a critical period may impede neural plasticity

Acknowledgments

This work was supported by NIH R01AG022101, Alzheimer Association IIRG-02-3758 as well as the American Heart Association pre-doctoral fellowship 0415162BB. We extend our thanks to Dr. Carl Flannery at Wyeth-Ayerst Laboratory for a gift of human recombinant ADAMTS4.

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