Changes in chondroitin sulphate proteoglycan in multiple sclerosis : A role for ADAMTS-9.

Multiple Sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) causing neurological disability in young adults, characterised by discrete acute and chronic lesions that are predominantly located in the CNS white matter. The role the extracellular matrix (ECM) has been widely studied as it is thought to be implicated in the pathogenesis of MS. Components such as chondroitin sulphate proteoglycans (CSPGs) are up-regulated in response to injury and inflammation and participate in the formation of sclerotic lesions. Research into the role of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) in CNS injury and inflammation has demonstrated that this protease may aid the recovery process by cleaving CSPGs, which are inhibitory to neurite outgrowth and axonal regeneration.In vivo studies were carried out investigating the distribution and expression of aggrecan and versican by dual label immunohistochemistry (IHC) and western blotting (WB) using antibodies which specially recognise cleavage-derived aggrecan and versican neoepitopes and intact protein. ADAMTS-9 expression was assessed by real time PCR (qRT-PCR), IHC and western blotting. In active lesions, both IHC and WB indicated that intact versican and aggrecan as well as their neoepitopes and ADAMTS-9 expression were all increased in areas of myelin thinning with ongoing demyelination and macrophage activation, compared to control and normal appearing white matter (NAWM) brain tissue. Immunostaining for CSPG neoepitope was particularly strong at the plaque border in chronic active lesions (CAL) compared to chronic inactive lesions (CL), NAWM and control brain tissue, suggesting active enzymatic cleavage of intact protein. IHC studies demonstrated that ADAMTS-9 was expressed predominantly by astrocytes, endothelium, macrophages and neurons with increased expression within MS active lesions. qRT-PCR 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.The role of CSPGs in neurite outgrowth from human neurons has been largely untested but is critical for understanding of regeneration following CNS injury. This thesis aimed to assess the effect of the ECM components, aggrecan, on neurite outgrowth of neuroblastoma cell line (SHSY-5Y) and expression of ADAMTS-9. This was achieved by several methods including qRT-PCR, ICC and WB. These data provided evidence that neurite outgrowth from SHSY-5Y cells is inhibited by aggrecan, this inhibition was associated with high level expression of ADAMTS-9.This thesis also aimed to investigate the expression of ADAMTS-9 by cells of the CNS to gain a better understanding of how these enzymes might be regulated and their possible role in the pathogenesis of MS. Cells were treated with pro-inflammatory cytokines in vitro, to mimic in vivo inflammatory conditions, and following this, ICC and WB data demonstrated that ADAMTS-9 was constitutively expressed by primary human astrocytes, microglia (CHME3) and neuronal (SHSY-5Y) cell lines in vitro, under basal condition. In primary astrocytes, ADAMTS-9 expression was increased following treatment with 10ng/mL interferon-y compared to control untreated cells. In contrast, dual treatment with interleukin-1 and tumour necrosis factor resulted in down regulation of ADAMTS-9. Thus illustrating the external inflammatory mileu influences the expression of ADAMTS-9 in lesions in MS, which in turn influences the degradation of the ECM components aggrecan and versican, which are also upregulated in lesions. Further work to understand how these changes in the ECM influence CNS repair is needed.