Although many neurological mechanisms that contribute to Alzheimer’s disease (AD) have been elucidated, none so far have yielded treatments that can improve AD patient prognosis or slow progression of the disease. Oxidative stress and inflammation in the brain have been identified as major hallmarks of AD, and studies suggest these phenomena induce neurodegeneration. However, connections between AD and the development of these symptoms remain poorly understood. The redox regulator Prx6 has been implicated by studies in humans and mice as a potential mediator between these phenomena. Prx6 may be able to influence oxidative stress and inflammation simultaneously using its peroxidase (PRX) and phospholipase (PLA2) enzymatic activities, respectively. Despite these findings, the roles of each Prx6 activity in AD progression have yet to be examined.
This thesis describes a body of experiments that suggest Prx6 may indeed contribute to the severity of AD, although the current evidence is insufficient to provide conclusive proof. Drosophila models of AD were crossed into lines carrying genomic constructs for over- or under-expression of the two fly Prx6 homologs: dPrx2540, which has strong PRX and PLA2 active-site homology with human Prx6, and dPrx6005, which retains homology only in its PRX v site. dPrx2540 and dPrx6005 protein expression in these lines was confirmed by crosses to Gal4 driver lines and subsequent western blotting. Lifespan experiments revealed that under expressing dPrx2540 in an amyloid-β background significantly reduces mortality. In contrast, over- or under-expression of dPrx6005 – which purportedly lacks PLA2 activity – caused significant but relatively minor changes in longevity. Additionally, locomotor tests suggest that dPrx2540 under-expression enhances activity in young and middle-aged AD flies. Recent work has focused on the creation of Drosophila lines in which all endogenous dPrx2540 copies are deleted using CRISPR-Cas9 methodology. These flies are being generated by designing and integrating vectors carrying Cas9 guide RNAs into the fly genome and expressing these RNAs coincidingly with germline Cas9. Mutant dPrx2540 transgenes with ablated peroxidase or phospholipase activities have also been constructed using PCR-based mutagenesis techniques. Vectors carrying these transgenes have been integrated into a separate group of flies. In the future, these flies will be combined into the dPrx2540-null background by crossing, and these CRISPR mutant dPrx2540 Drosophila lines will be further combined into an AD background. This will allow lifespan assays and other measurements of AD phenotypes to be performed, which will provide a more rigorous analysis of the specific roles of Prx6’s two enzymatic activities in AD.
Biological Sciences, General
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Drolet, Nathan A., "Peroxiredoxin 6 and Phospholipids in Alzheimer's Disease" (2019). Biological Sciences Theses and Dissertations. 4.