Abstract
Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.
Highlights
Alzheimer’s disease (AD) is nowadays responsible by 50 to 80 percent of dementia cases [1] and is mainly characterized by two types of lesions: neurofibrillary tangles (NFTs) and neuritic plaques
L55P TTR, suggesting that TTR amyloidogenecity influences its binding to the peptide; at the level of are mainly constituted by beta-amyloid (A-Beta) aggregation, we did not detect any differences between TTR mutants, as both non-amyloidogenic and amyloidogenic variants were able to prevent A-Beta fibrilization with approximately the same extent, as evaluated by transmission electron microscopy (TEM) [6]
We further compared if different TTR variants differently affect A-Beta fibrillogenesis, making use of a dot blot assay with the A11 antibody, described as specific for oligomeric species
Summary
Alzheimer’s disease (AD) is nowadays responsible by 50 to 80 percent of dementia cases [1] and is mainly characterized by two types of lesions: neurofibrillary tangles (NFTs) and neuritic plaques. Neuritic plaques are extracellular amyloid deposits found in the brain and are mainly constituted by beta-amyloid (A-Beta) peptide. Accumulation of ABeta is due to disregulated proteolytic processing of its precursor, the Amyloid Precursor Protein (APP). Schwarzman et al described that normal cerebrospinal fluid (CSF) inhibits amyloid formation [4] and concluded that transthyretin (TTR) was the major A-Beta binding protein in CSF, leading to a decrease in the aggregation state of the peptide [4]. Our group characterized the TTR/A-Beta interaction showing that TTR is capable of interfering with A-Beta fibrilization by both inhibiting and disrupting fibril formation [6]. A-Beta new peptides (1–14) and (15–42), generated upon cleavage by TTR, showed lower amyloidogenic potential than the full length counterpart [7]
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