Expression of ADAMTS-9 in the Central Nervous System in Multiple Sclerosis and Its Relationship to Cleavage of Aggrecan and Versican

A Disintegrin And Metalloproteinase with Thrombospondin motifs (ADAMTSs) are proteases which are able to cleave aggregating chondroitin sulphate proteoglycans (CSPGs), including aggrecan and versican. Extracellular matrix (ECM) alterations have been reported in the central nervous system (CNS) in multiple sclerosis (MS) in post mortem white matter tissue, due to both the release and activation of matrix metalloproteinases (MMPs) and ADAMTSs as well as increased synthesis of ECM components. To elucidate the potential pathophysiological role of ADAMTS-9 in ECM changes in MS, we investigated expression of aggrecan and versican by dual label immunohistochemistry (IHC) and western blotting (WB) using antibodies which specifically recognise cleavage-derived aggrecan and versican neoepitopes or intact proteins. ADAMTS-9 expression was assessed by real-time RT-PCR (qPCR), IHC and WB. Snap frozen blocks (40) of brain tissue were obtained from the UK MS Society Tissue Bank, Imperial College London. Blocks were classified by routine histology using haematoxylin & eosin and Oil Red O to assess inflammation and demyelination respectively and by IHC using antibodies against human leukocyte antigen (HLA-DR), to assess macrophage activation and myelin oligodendrocyte glycoprotein (MOG) for evidence of demyelination. Ten blocks each, of normal appearing white matter (NAWM), active lesions (centre and border) and control white matter were analysed. IHC staining was quantified using Image J software. WB quantitation of bands was performed using the LI-COR instrument software, normalising to a beta actin loading control. In active MS lesions, both IHC and WB indicated that intact versican and aggrecan as well as their neoepitopes and ADAMTS-9 expression were increased in areas of macrophage activation, compared with in NAWM. Immunostaining for CSPG neoepitopes was particularly strong at the plaque border, suggesting active enzymatic cleavage of intact protein. IHC studies demonstrated that ADAMTS-9 was expressed predominantly by macrophages and neurons with increased expression within MS lesions. Preliminary qPCR studies confirmed ADAMTS-9 expression in MS tissue. In summary, this study provides evidence that ADAMTS-9 plays an important role in cleavage of the ECM CSPGs, aggrecan and versican, in active lesions in MS. Further work will assess the functional consequence of these changes in the ECM using in vitro neurite outgrowth assays.