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Title: Altered heparan sulfate in ageing and dementia : a potential axis for the dysregulation of BACE-1 in Alzheimer's disease
Author: Clarke, H. E.
ISNI:       0000 0004 6425 224X
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2017
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Alzheimer’s disease (AD) is characterised by amyloid plaques composed of amyloid-beta (Aβ), the cleavage product of the amyloid precursor protein (APP) by the protease beta-secretase (BACE- 1). Heparan sulfate (HS) inhibits BACE-1 and holds potential as a new drug discovery target; in vivo HS may act as a brake on the generation of Aß via regulation of BACE-1. Previous work has identified the sulfate moieties in HS as key determinants in the efficacy of BACE-1 inhibition. Structural changes in HS are known to occur with ageing and we hypothesised that these changes could result in reduced BACE-1 inhibition and ultimately elevated production of Aβ. Strong anion exchange chromatography was used to assess disaccharide composition of HS from AD (n=20) and age-matched control (n=15) brain tissue. TaqMan® array profiling of HS-related genes was also carried out to explore expression levels of HS-related genes that may be responsible for downstream HS structural changes. HS purified from AD and age-matched control samples was assessed for its ability to inhibit BACE-1 using FRET-based BACE-1 activity assays and finally, manipulation of endogenous HS in HEKSweAPP cells with RNAi was carried out to explore the possibility of modulating generation of the toxic Aβ species. HS from AD tissue was found to carry a significantly decreased proportion of the di-sulfated ΔUA-GlcNS(6S) disaccharide vs. controls (p < 0.01) and increased levels of the lesser-sulfated ΔUAGlcNAc( 6S) unit vs. controls (p < 0.05). Furthermore, significantly more total HS was present within control brain tissue (122.3μg/100mg) vs. AD (78.6μg/100mg) (p < 0.01). TaqMan® array analysis revealed significant alteration in expression of HS biosynthetic genes with AD including upregulation of HS6ST1 (p < 0.05) and a strong trend for down regulation of HS6ST3, coupled with up regulation of SULF1. These changes may go some way to explain changes in the level of sulfation of HS particularly, 6-O sulfation, as observed by structural analysis. Most noticeably, BACE-1 activity assays revealed a significant reduction of BACE-1 inhibition efficacy by HS from AD patients (p < 0.05). In addition, knockdown of SULF1 in HEKSweAPP cells, which would be expected to elevate 6-O sulfation, generated a significant reduction in Aß. Our observation that AD brain HS contains fewer di-sulfated ΔUA-GlcNS(6S) disaccharides, alongside observed upstream gene expression changes, would be consistent with a less sulfated HS chain with reduced ability to inhibit BACE-1 thus generating more Aß as observed in AD. The observed reduction in BACE-1 inhibition efficacy by HS with AD confirms our hypothesis that structural changes in HS may contribute to modulating AD pathogenesis in patients. Finally, these studies support the idea that HS-based therapeutics might provide the basis for novel disease modifying drugs that could prove beneficial in future efforts to treat an underlying cause of AD.
Supervisor: Turnbull, J. E. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral