Neurotoxic aggregates of amyloid beta (Aβ) peptide contribute to the etiology of Alzheimer's disease (AD). Aβ1-42 forms oligomeric structures that undergo further aggregation into protofibrils and fibrils. Oligomeric Aβ1-42 is more toxic than monomers or mature fibrils. In this work, we used two distinct approaches to inhibit Aβ1-42 oligomerization and toxicity. First, seven distinct but overlapping Aβ fragments were used to identify their individual aggregation propensities and their effects on Aβ1-42 oligomerization and cytotoxicity. Studies on suppression of Aβ1-42 cytotoxicity by peptides, including those derived from Aβ1-42, have been conducted before, but peptides encompassing the whole Aβ1-42 sequence have not been systematically analyzed. Aβ1-42 was allowed to aggregate and form oligomeric assemblies in aqueous buffer for 4 h in the absence or presence of 2-fold molar excess of an Aβ fragment. Cytotoxicity analysis then recorded the impact of each fragment on Aβ1-42 cytotoxicity as well as the toxicity of the fragments themselves. An enzyme-linked immunosorbent assay that detects oligomeric Aβwas used to determine the effect of each fragment on Aβ1-42 oligomerization after 4 h of aggregation. Four fragments of Aβ1-42 inhibited the toxicity of oligomeric Aβ1-42 to various degrees, while two others conferred no cellular protection against Aβ1-42 toxicity. Interestingly, one fragment enhanced Aβ1-42 toxicity after 4 h of aggregation. Three of the four fragments that blocked Aβ1-42 toxicity partially disrupted oligomer formation, showing correlation between the inhibition of Aβ1-42 aggregation and the inhibition of cellular toxicity. Second, we examined whether protein disulfide isomerase (PDI), a chaperone mainly found in the endoplasmic reticulum, could reverse the oligomeric state of aggregated Aβ1-42 and thus its toxicity. Previous work has demonstrated that PDI inhibits Aβ1-42 aggregation at sub-stoichiometric concentrations. To assess PDI's effect on Aβ1-42 toxicity, Aβ1-42 was allowed to aggregate for 2 h before the addition of PDI at a 1:10 molar ratio of PDI to Aβ1-42 and then allowed to aggregate for another 2 h. MTS cytotoxicity assays using PC-12 cells showed that adding PDI 2 h after the start of aggregation improves cell survival. Through a differential centrifugation assay followed by Western blot, we qualitatively illustrated that PDI can reverse a 2 h aggregate of Aβ1-42 to the monomeric state. Overall, in this project we have learned that inhibiting the oligomeric assembly of Aβ1-42 directly decreases the effect of Aβ1-42 toxicity. Inhibition of Aβ1-42 toxicity was seen with both fragments derived from Aβ1-42 and PDI, shedding light into two novel approaches as possible therapeutics for AD.
| Identifer | oai:union.ndltd.org:ucf.edu/oai:stars.library.ucf.edu:etd2023-1263 |
| Date | 01 January 2024 |
| Creators | Zabala Rodriguez, Maria C |
| Publisher | STARS |
| Source Sets | University of Central Florida |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Thesis and Dissertation 2023-2024 |
| Rights | In copyright |
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