Alzheimer’s disease (AD) is a chronic, progressive, neurodegenerative disorder of the brain, and has been characterised by the presence of amyloid plaques (APs) and neurofibrillary tangles (NFTs). According to the amyloid cascade hypothesis (ACH), amyloid beta (Aβ), an erroneous metabolite of amyloid precursor protein (APP) leads to the formation of both soluble and insoluble forms of aggregates that eventually deposit as APs. The soluble Aβ aggregates are the major cellular destructive species in cholinergic regions of the brain. Currently available AD treatments such as cholinesterase inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists offer only temporary benefits to patients. By focusing on the underlying pathogenesis of AD, several ACH based disease-modifying treatment strategies with the possibility of controlling the progression of the disease are now under investigation. The potential anti-amyloid strategic targets are (a) decreasing Aβ production; (b) inhibiting Aβ aggregation; (c) targeting pathological chaperones that interact with Aβ; (d) altering the aggregation pathways to prevent formation of toxic Aβ aggregates; and (e) clearance of Aβ by immunotherapeutic and enzymatic means.
To develop anti-amyloid strategies, it is vital to understand the roles of extrinsic and intrinsic factors in Aβ aggregation. Important extrinsic factors are pH, ionic strength, and the sulfated carbohydrate element, glycosaminoglycans (GAGs). In particular, GAGs have received much attention because of their ability to enhance Aβ aggregation and protect fibrils from proteolysis. Typically, Aβ40/42 has been used in anti-amyloid studies; Aβ28 is sometimes used because of its low cost, ease of synthesis, and relatively facile preparation for biophysical and biological experiments. The question of whether Aβ28 is an appropriate surrogate for Aβ40/42 in drug discovery assays has been posed. To answer this question, chapter two of this thesis examines the suitability of Aβ28 for drug discovery assays, specifically the role of extrinsic factors on Aβ28 aggregation using biophysical techniques such as thioflavin T (ThT) assay, circular dichroism spectroscopy (CD), transmission electron microscopy (TEM) and zeta potential (ZP). The aggregation propensity of Aβ28 was shown to resemble that of Aβ40/42 in relation to the effect of ionic strength and GAGs. However, Aβ28 proved unsuitable for AD drug discovery assays because Aβ28 aggregates only near its isoelectric point (pH ~5.5), conditions which are not physiologically relevant.
The third chapter of this thesis explains the effect of nutritional supplements, homotaurine and taurine, on Aβ40 aggregation. Information related to the effect of these compounds on Aβ aggregation in the presence of extrinsic GAGs, and indeed evidence to support their classification as GAG mimetics, is lacking. To address these issues, this study examined the effect of homotaurine and taurine on Aβ40 aggregation, in the presence and absence of GAG, using biophysical methods and in vitro cell-based assays. The well known anti-amyloid properties of polyphenol compounds such as curcumin, resveratrol, epigallocatechin gallate (EGCG) and tannic acid were included as reference compounds. It was confirmed that taurine and homotaurine are GAG mimetics that exhibit weak inhibition of heparin facilitated aggregation in the ThT assay. Homotaurine showed a stronger inhibition of mature fibril formation than taurine under TEM. Curcumin and resveratrol were omitted to remove analytical bias in the assays. The GAG mimetics and EGCG and tannic acid were shown, using CD, to inhibit the conformational transition to beta sheet structure. Furthermore, EGCG, homotaurine and taurine were similarly neuroprotective in primary neuronal cell culture whereas tannic acid was inherently toxic.
The anti-amyloidogenic and neuroprotective properties of the GAG mimetic eprodisate (a di-sulfonate) and novel analogues of eprodisate (mono-, tri-, tetra- & hexa-sulfonates) were investigated against Aβ40 aggregation and these studies are described in the fourth chapter of this thesis. Eprodisate is a GAG mimetic clinical trial drug for the treatment of amyloidosis, a disease caused by the aggregation of serum amyloid A protein (SAA). To further understand the role of eprodisate and its analogues on Aβ40 aggregation, mono- and di-sulfonates (eprodisate) were commercially obtained, whilst three other compounds, namely the tri-, tetra- and hexa-sulfonates were synthesised. Anti-amyloidogenic studies were performed, investigating the structure-activity relationships between the number of sulfonate groups and the inhibition of Aβ40 aggregation. Furthermore, mouse primary neuronal cells were used to study the neuroprotective effects of the lead compounds, eprodisate and tetra-sulfonate. It was found that eprodisate (a di-sulfonate) inhibits mature fibril formation but did not inhibit the early stage of Aβ40 aggregation. The tetra-sulfonate exhibited potent GAG mimetic activity by inhibiting Aβ40 aggregation at both early and mature fibril stages. Both eprodisate and the tetra-sulfonate possessed neuroprotective effects.
Homotaurine has recently been withdrawn from clinical trials. However, the studies reported in this thesis have shown conclusively that homotaurine, taurine, eprodisate and the tetra-sulfonate compound all inhibit Aβ40 aggregation in the presence of GAG (heparin sulfate). This observation strongly supports the GAG-mimetic mechanism for these compounds. It is believed that such studies will assist in the development of promising anti-AD drug candidates.