The Role of Fibrates and Soluble Amyloid Precursor Alpha in Alzheimer’s Disease and Longevity

Owens, Lauren and Parkin, Edward and Benedetto, Alexandre and Gaffney, Christopher and Dawson, Neil (2023) The Role of Fibrates and Soluble Amyloid Precursor Alpha in Alzheimer’s Disease and Longevity. PhD thesis, Lancaster University.

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Abstract

The amyloid cascade hypothesis and associated accumulation of neurotoxic amyloid beta (Aβ)-peptides has been the central causation theory underlying the neurodegenerative condition Alzheimer’s disease (AD) for many years. Whilst these peptides are derived from the amyloid precursor protein (APP) far less research has focused on the fact that A Disintegrin And Metalloprotease 10 (ADAM10) mediated non-amyloidogenic proteolysis of the same protein can also yield a beneficial soluble APP alpha (sAPPα) fragment. As such, upregulation of this latter proteolytic event is an area of research that has, more recently, started to gain traction as a disease therapy for AD. A group of drugs known as fibrates have previously been shown to upregulate ADAM10 through peroxisome proliferator-activated receptor α (PPARα), reducing amyloid plaque load and improving learning and memory in AD mouse models. In the current study we have investigated the effects of fibrates on APP cell biology in AD-relevant SH-SY5Y neuroblastoma cell lines and on life span in C. elegans. Furthermore, we present design and implementation data pertaining to the development of neuron-specific inducible plasmid expression systems for the elevation of protective proteins such as sAPPα in cell systems and, potentially, as gene therapies. Our results show that two fibrates, gemfibrozil and bezafibrate, had few significant effects on APP proteolysis in four AD-relevant SH-SY5Y cell lines. Regarding C. elegans, neither fibrate produced a consistent increase in lifespan, although 1 μM gemfibrozil increased lifespan in worms fed inactivated bacteria. To elucidate the mechanism, and whether the C. elegans ADAM10 homolog, SUP-17, was involved, we examined healthspan, mitochondrial networks, intestinal lipid content, gut pathology, and screened genes for their involvement in the mechanism of action of gemfibrozil. We found that SUP-17 was not involved; instead, fatty acid desaturation was increased through the PPARα homolog, NHR-49. During our fibrate studies in the SH-SY5Y cell lines, we noticed that the ADAM inhibitor, batimastat, did not decrease secretion of non-amyloidogenically-derived sAPP in SH-SY5Y cells overexpressing the APP β-secretase (β-site APP cleaving enzyme 1, BACE1). Further investigation of APP C-terminal fragments in the four cell lines, in addition to studies using the BACE inhibitor, β-secretase inhibitor IV, revealed that the non-amyloidogenically-derived fragment in these cells was sAPPβ’, produced by an alternative BACE1-mediated cleavage of APP. A decrease in the sAPPα/sAPPβ’ axis was found to be responsible for decreased viability in SH-SY5Y-BACE1 cells, a phenomenon that was reversed by transfection with sAPPα. This provided rationale for directly enhancing sAPPα as a treatment for AD. As such, we designed a dual-plasmid (regulator and response plasmids) system for the inducible, neuron-specific expression of sAPPα and a second therapeutic protein, VPS35, and described how it could, in the future, be combined into a single piggyBac plasmid. The experimental generation of versions of the plasmids required to test inducibility, neuron-specificity, and protein expression is described, and several cell lines expressing the regulator plasmid, as well as one expressing the response plasmid and facilitating the induction of sAPPα expression, have been generated. To conclude, gemfibrozil and bezafibrate did not enhance ADAM10 expression or promote non-amyloidogenic APP processing in our investigations. However, we have partially elucidated a pathway that mediates longevity in C. elegans following low-dose gemfibrozil treatment. In terms of AD, we are now focusing on developing cell lines that express sAPPα and VPS35 in response to activator ligands, such as doxycycline, with a view to examining the effect of therapeutic protein expression on processes such as cell proliferation and viability.